WO1996036728A1 - Fluorescence ratio scanning - Google Patents

Abstract

The present invention provides methods and an apparatus for fluorescence ratio scanning which may be used to evaluate the stability of cellular and cell-free proteins when subjected to a stress.

Description

FLUORESCENCE RATIO SCANNING

FIELD OF THE INVENTION

The present invention provides methods and an apparatus for fluorescence ratio scanning.

BACKGROUND OF THE INVENTION

While a substantial fraction of the modern scientific attack on the problem of clinical treatment of malignant tumors centers on understanding the abnormal genetics of these tissues, there is still a significant effort devoted to preferentially destabilizing the physiology of tumors through a combination of locally applied physical and chemical stresses, such as chemical agents. Commonly recognized physical stresses include ionizing radiation and temperature variation, such as hyperthermia. Chemical stresses include chemotherapeutic compounds which have a wide range of actions, many of which are poorly understood, but which include alkylation of DNA, anti-folate activity, fraudulent DNA base substitution, various methods of inhibiting DNA synthesis, and hormonal impact on the malignantly transformed target tissue. Although the present invention focuses on the effects of temperature, of which, hyperthermia has been extensively investigated since the 1960's, one of ordinary skill will appreciate the disclosed method and apparatus finds broad application in evaluating the extent of protein destabilization upon application of an external stress.

Hyperthermia involves local warming of target tissue to temperatures between 40°C and 45°C, generally in combination with other agents, such as those mentioned above. The generally accepted cause of increased mortality at elevated temperatures is protein denaturation, and this is the underlying mechanism justifying pursuit of treatments of this type.

While some believe hyperthermic treatment shows significant promise, the applicants believe the following two factors prevent it from being as powerful a treatment as hypothermia. First, the clinically accessible temperature range overlaps the range of normal fevers in mammals. Even cancer cells have a naturally evolved response to the stress, through heat shock proteins, which are known to facilitate protein refolding upon return to normal physiological conditions. In non-hibernating mammals this response is not initiated at low temperatures. Perhaps just as important is the nature of the stability (free energy) vs. temperature curve for most cellular proteins. In general, most cellular proteins are quite stable at 37°C, although in the strictly thermodynamic sense they may be optionally stabilized under conditions considerably different from those of the intracellular medium. Nevertheless, the fact that proteins are reasonably stable in vivo, and that the stability is high over the 37°C to 45°C temperature range implies that most of the proteins are still reasonably stable and, presumably, functional at 45°C.

In contrast, the present disclosure demonstrates that at or very near 0°C most aqueous phase or water soluble proteins, and perhaps membrane proteins as well, are expected to undergo very significant destabilization. This discovery suggests that the majority of proteins, when rewarmed to 37°C, will remain significantly destabilized with respect to their initial state prior to chilling.

Although not linking their results to protein destabilization, Fay and Henny, in 1938, reduced tumor sizes and pain relief following local application of cold (4.4°C to 10°C). Moreover, in 1916, Simpson showed that skin exposed to ice packs showed a significant increased sensitivity to X-Rays. Mandrik, in 1959, reported that cooling of whole rabbits with Brown-Pearce carcinoma led to an inhibition in the number of subsequent metastases when compared to uncooled control animals. Mincheff et al. have recently shown that cultured T-cells exposed to gamma radiation followed by exposure to 22°C or 0°C showed increasing loss of co-stimulating activity as the time of exposure to 0°C increased. Cells showed greater than 70% loss of activity with respect to 22°C at the highest radiation doses and one hour exposure to 0°C. (Mincheff, M. et al., Vox Sanguinis, 65, 18, 1993).

Any suggestion that these results are due to protein destabilization seems to contradict the prevailing dogma that proteins are stabilized at temperatures near 0°C, and that, additionally, many proteins are resistant to freezing. The explanation for the seeming contradiction is two-fold. In the first case, workers have generally failed to investigate loss of activity after exposure to temperatures near 0°C. Recent reports

(Baracca, A. et al. Biochim. Biophys. Acta 976, 77, 1989;

Tsunekawa, S. et al. Transplantation, 52, 6,999, 1991;

Harding, C. and Unanue E. Eur. J. Immunol., 20, 323, 1990; Sassenrath, G. et al. Arch. Biochem. Biophys., 282, 2, 302, 1990; Nakanishi, Y. et al. J. Biochem. 115, 328, 1994) however, show just this loss of activity in several enzymes near 0°C.

In the case of freezing resistance, in fact, most proteins from organisms not resistant

to freezing, when frozen without stabilizers such as salts, polyols and amino acids show some loss of biological activity (Tamiya, T. et al. Cryobiology, 22, 446, 1985; Carpenter, J. and Crowe J. Cryobiology, 25, 244, 1988; Bergh, F. et al. Anal. Biochem. 193, 287, 1991). For many proteins this loss of activity is at least in the range of 10-25%, which is consistent with the level of denaturation in these systems reported by the present method. The destabilization exemplified herein is believed to persist for hours, and, for some proteins, for days. Hypothermia, therefore, is believed to be a much more effective way of inducing protein instability in localized malignant tumors with a higher probability of potentiation when combined with chemotherapeutic agents, radiation or other concomitant stressor subsequent to rewarming.

The present invention provides methods and apparatus for evaluating the onset, duration and extent of protein destabilization in solution, including protein destabilization in cells, vesicles or other multiphase mixtures.

Work on the thermodynamics of protein denaturation dates back more than half a century to studies on the rate of albumin denaturation as a function of urea concentration and temperature. In the last 25 years a growing body of data from microcalorimetry, optical rotation and fluorescence ratio measurements has contributed to a widely accepted standard thermodynamic description of protein unfolding. In the standard model, the denaturational increment of heat capacity is taken to be a positive constant, which leads immediately to the assumption that the excess enthalpy of denaturation is a linearly increasing function of temperature. The Gibbs free energy is also determined from the assumption of a positive constant heat capacity increment to be a linear plus linear times logarithmic function of temperature, convex in the positive (free energy) direction with one maximum and two zeroes (Privalov, P. Adv. Protein Chem. 167, 1979; Privalov, P. Annu. Rev. Biophys. Chem. 18, 47, 1989).

Taken together, these assumptions produce a well defined set of predictions about what will actually be observed when these thermodynamic variables are measured experimentally. In the range of about 20°C to 80°C the predictions of the generally accepted theory seem to be in moderately good agreement with differential scanning calorimetry (DSC) experiments. However, the fundamental assumption of a constant denaturational heat capacity increment is known to be wrong at both very high and very low temperatures. At extremely low temperatures, ΔC_p must approach zero according to the third law of thermodynamics, while at higher subambient temperatures it may be either positive or negative depending upon the detailed interaction between protein and solvent as a function of various conformational states of the protein. At temperatures somewhat above the high temperature denaturation point, the heat capacity could be expected to increase as the difference in free volume between the unfolded and folded configurations increase (s) (Hatley, R.M. and Franks, F., Faraday Discuss., 93, 249, 1992). It has been argued that at very high temperatures the heat capacity should decrease because the newly exposed hydrophobic surfaces of unfolded protein become progressively less able to order vicinal water as the temperature increases. In any event, if it can be shown that either the heat capacity or the enthalpy of protein unfolding are significantly non-linear in a given temperature range, then the constant heat capacity assumption is seriously in error.

The consequences of a nonconstant heat capacity increment have been examined in detail only for a linear variation of ΔC_p by Franks et al . Biophys. Chem, 31, 307, 1988. More recently, Wintrode P. et al. Proteins: Structure, Function and Genetics, 18, 246, 1994 have acknowledged that the denaturational heat capacity increment is non-linear and Franks et al Biophys. Chem, 31, 307, 1988 have also suggested that the published heat capacity data are at least quadratic. The applicants are aware of no investigation wherein the consequences of the assumption that ΔCp is quadratic has been explored as has Franks' assumption of linear dependence of ΔC_p on temperature.

SUMMARY OF THE INVENTION

The present invention provides a method of measuring protein stability including the onset, duration and degree of instability. The method of the present invention can be used to determine protein stability or instability produced by application of external stresses, forces or agents. It is therefore, an object of the present invention to provide a method for evaluating agents for affecting, for example, enhancing or limiting, protein stability or instability.

It is another object of the present invention to provide a method for evaluating agents for enhancing cellular stability or instability based on the ability of the agent to stabilize or destabilize cellular proteins.

In one embodiment of the present invention, a protein formulation either in solution, frozen solution, or lyophilized form is evaluated by fluorescence ratio scanning as to the amount of destabilization, or unfolding, which is produced when the protein is subjected to a constant stress or externally applied agent (s) and the temperature is systematically increased or decreased from a reference temperature.

The disclosed method of evaluating agents for enhancing the stability of protein in a protein formulation comprises the steps of providing a first sample of the protein formulation of interest; determining a range of temperature, at a constant stress, over which the protein contained in the formulation remains stable; and measuring the fluorescence emission spectrum of the protein formulation in a range of 300 - 500 nm when excited by a light beam in a range of 270-700, preferably 270-290 or 500-700, nm, in the range of temperature determined above, to provide a first spectrum.

A second sample of the protein formulation is provided wherein the protein of the formulation is heat denatured, at the constant stress, and the fluorescence emission spectrum of this sample, at constant stress, is measured in a range of 300-500 nm when excited by a light beam in a range of 270-700 nm to provide a second spectrum. The first and second spectra are compared to determine lambda (1) and lambda (2), wherein lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between the first and second spectra.

A third sample of the protein formulation is provided wherein the protein of the formulation is maximally stabilized, at the constant stress, to provide a stable base line by repeatedly recording the fluorescence intensity at lambda (1) and a fluorescence intensity at lambda (2) when the sample is excited with light of a wavelength of 270-700 nm to form a ratio of fluorescence intensity at lambda (1) divided by a fluorescence intensity at lambda (2), while simultaneously varying or ramping the temperature over the range of temperatures as determined above. The stable base line is the ratio over the temperature range.

A fourth sample of the protein formulation is provided wherein the protein has been heat inactivated at a temperature, T, at a constant stress, such that the protein is fully destabilized over a temperature range of interest, to provide a scaling sample wherein the protein is wholly inactive but not necessarily fully denatured. The fluorescence of the fourth sample is scanned with an excitation light beam in the range of 270-700 nm while simultaneously cooling the sample from a temperature T where the protein is maximally destabilized, to a lower temperature of interest, to provide an unstable base line by repeatedly recording the fluorescence intensity at lambda (1) and a fluorescence intensity at lambda (2) to form the ratio of fluorescence intensity at lambda (1) divided by the fluorescence intensity at lambda (2). The unstable base line is the ratio over the temperature range.

A fifth sample of the protein formulation is provided as a control sample. The fluorescence scan of the fifth sample is performed as follows, to provide a standard experimental ratio curve: the fluorescence intensity at lambda (1) and the fluorescence intensity at lambda (2) are repeatedly recorded when the sample, at constant stress, is excited with a light beam in the range of 270-700 nm, and used to calculate the ratio of the fluorescence intensity at lambda (1) divided by the fluorescence intensity at lambda (2). The fluorescence measurements of the fifth sample are determined while scanning or ramping the temperature over a temperature range of interest.

A sixth sample of the protein formulation is provided which contains an agent to be tested as a stabilizer. The sixth sample will provide an experimental ratio curve comprised of a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2), while the temperature is simultaneously scanned over the temperature range of interest.

The ability of any test agent to enhance stability of a protein in a protein formulation is evaluated by comparing the recorded ratios of the fifth sample (control) and the test plurality of ratios of the sixth sample (test), such that for any given temperature, stability will be indicated as having been increased whenever the value of the difference in ratio between the unstable base line and the test ratio curve is larger than the value of the difference in the ratio of the unstable base line and the ratio of the control curve, and given that the differences are of the same sign.

The efficiency of any agent to stabilize a protein in a protein formulation can be quantitated by dividing, for any given temperature, the value of the control ratio curve minus the value of the test ratio curve, by the value of the unstable base line ratio minus the value of the stable base line ratio to yield an estimate of the fraction protein stabilized.

In another embodiment of the present invention, a method of evaluating cellular destabilization or stabilization agents is provided. In this embodiment, the extent of protein destabilization or stabilization is measured in whole cells, as opposed to proteins in solution, in a manner similar to that described above. Specifically, the present invention provides a method for evaluating agents for enhancing cellular instability by: (a) providing a first sample of cells of a single or homogeneous cell type;

(b) determining a range of stress over which the cells are viable;

(c) measuring within the range of viability the fluorescence emission spectra of the cells in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a first spectrum;

(d) providing a second sample of the cells of the single cell type wherein the proteins of the cells are substantially denatured;

(e) measuring the fluorescence emission spectra of the cells of the second sample in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a second spectrum;

(f) comparing the first and second spectra to determine lambda (1) and lambda (2), such that lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between the first and second spectra;

(g) providing a third sample of the cells of the single cell type;

(h) determining a control plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in the third sample over a range of increasing stress followed by a range of decreasing stress, wherein the increasing stress produces protein instability; (i) providing a fourth sample of the cells of a single cell type;

(j) adding the agent to the fourth sample to produce a test sample;

(k) determining a test plurality of ratios of fluorescence intensity of lambda (1) to fluorescence intensity in the test sample over a range of increasing stress followed by a range of decreasing stress, as in step (h) ; and

(1) evaluating the ability of the agent to enhance cellular instability by comparing the control and the test plurality of ratios, such that instability will be increased where the control plurality of ratios is greater than the test plurality of ratios for any given amount of stress in at least one of the increasing or decreasing directions.

It is another object of the present invention to provide a fluorescence ratio scanning apparatus.

In one embodiment of the invention, an apparatus is provided for determining the effect of stress on cellular proteins which comprises:light emitting means for emitting a beam of light at a desired wavelength of light; means for focusing the beam onto the cell culture sample; means for scanning the beam in two dimensions across said cell culture; means for changing ambient temperature of the cell culture; means for calculating the transmitted light through the cell culture; and means for calculating an intensity ratio of two wavelengths of back-scattered fluorescent light from the sample, wherein the ratio provides information indicative of the state of the cell culture; and means for calculating the intensity of backscattered excitation light.

The light emitting means of the apparatus is preferably a laser. The desired wavelength is preferably between 500 nm and 700 nm. The ratio of the apparatus of the present invention is calculated using the following condition (1):

where Λ₀ is the absolute fluorescence intensity of a folded protein, Λ₁ is the absolute fluorescence intensity of a fully unfolded protein, and Λ is the measured fluorescence intensity of a mixture of the folded and unfolded forms at wavelengths λ₁ and λ₂.

The apparatus of the present invention further comprising a Fourier photomultiplier tube disposed so as to measure the back-scattered light and a proportional photomultiplier so as to measure the transmitted light through the cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a. shows a best fit to the fluorimetrically derived free energy data using the equation developed in this invention. The r² is >.99943 over the temperature range -15 to 10°C and 65 to 95°C inclusive. At low temperatures, the renaturation is associated with a relatively small molar enthalpy change and a modest net shift in the proportions of native and denatured states, so the net observable enthalpy is predicted to be small, i.e., about 20 kJ/mole. Curve 2 of the insert shows the raw experimental curve of fluorescence ratio for β lactoglobulin 0.1M NaCl pH2. Curve 1 of the insert represents the raw fluorescence ratio for β-lactoglobulin in 5 nM sodium sulfate used to extrapolate the stable base line of native state protein stability. Curve 3 of the insert shows the unstable base line (heat denatured state) for β-lactoglobulin.

Figure 1b. Best fit of classical constant ΔC_p model to the applicants' fluorimetric data forβ-lactoglobin. The r² for fit of the low noise high temperature data between 69°C and 89°C is >0.9915 for a ΔC_p of 6.5 kJ/mole°C, in excellent agreement with published calorimetric data. The predicted observable ΔHden is about 267 kJ/mole, also in close agreement with the applicants' experimental calorimetric results and those of others (Griko, Y. and Privalov, P., Biochemistry, 31, 8810, 1992). The complete failure of this classical constant model to predict low temperature behavior is noted, however.

Figure 2a. Effect of pH and temperature on β- lactoglobulin unfolding as measured by the fluorescent ratio technique. Fluorescence emission ratio I₃₅₀ /I₃₃₀ nm was measured as a function of temperature, for a given pH. The pH was varied from 2 to 13 with the traces representing, measurements at pH 2, 8, 9, 10, 10.5, 11 11.5, 12, 12.5 and 13.

Figure 2b. Effect of pH and temperature on β- lactoglobulin unfolding for pH's 2, 5.8, 7, 7.5 and 7.8. Figure 3a. Effects of various concentrations of the denaturant urea on cold and heat denaturation of β-lactoglobulin in 0.1 M NaCl pH2. If low concentrations of urea are used at temperatures near 0°C, the reversal of denaturation occurs at a lower subzero temperature, indicating an increasing contribution of stabilization as the temperature is lowered and providing additional evidence that this is not oligomerization of the native state. The insert details the low temperature denaturation-renaturation events. The straight line is the base line of maximal stability extrapolated from the fluorescence ratio data of β-lactoglobulin in a solution of 0.005M Na₂SO₄ pH2 using the temperature range 25-65°C and extended for the entire temperature range of investigation.

Figure 3b. Influence of various concentrations of the denaturant guanidine hydrochloride on cold and heat denaturation of β-lactoglobulin in 0.1M NaCl pH2. The insert top to bottom details the low temperature denaturation-renaturation events. The straight line is the base line of maximal stability extrapolated from the fluorescence ratio data of β-lactoglobulin in a solution of 0.005M Na₂SO₄ pH2 using the temperature range 25-65°C and extended for the entire temperature range of investigation.

Figure 4a. Unfolding-refolding behavior of β- lactoglobulin during slow cooling followed by slow freezing (0.3°C/min) in concentrated urea solutions containing 0.1M NaCl pH2. The change in protein structure is monitored by wavelength shift of peak fluorescence intensity here because standard spectrofluorimetric devices are unable to record an accurate ratio in frozen solution.

Figure 4b. Unfolding-refolding behavior of β- lactoglobulin as a result of slow reheating and thawing

(0.3°C/min) in concentrated urea solutions containing 0.1M

NaCl pH2, immediately after slow freezing (Fig. 4a). The change in protein structure was also measured by peak wavelength shift.

Figure 5a. Temperature dependant effects of various levels of stabilizing electrolytes (0.025, 0.05, 0.1, 0.2, 1.0, 2.0 M NaCl, 0.1 M Na₂HPO₄ and 0.005 M Na₂SO₄) at pH2, on the fluorescence ratio ofβ-lactoglobulin. The insert top to bottom details the low temperature denaturation-renaturation events. The straight line is the base line of maximal stability extrapolated from the fluorescence ratio data of β-lactoglobulin in a solution of 0.005M Na₂SO₄ pH2 using the temperature range 25-65° C and extended for the entire temperature range of investigation.

Figure 5b. Fluorescence ratio data showing the effects of the stabilizer glycerol in concentrations 0, 0.25 M, 0.5 M, 1.0 M, 2.0 M 5.0 M and presence of 0.1M NaCl at pH2.on β-lactoglobulin thermal behavior. The insert top to bottom details the low temperature denaturation-renaturation events. The straight line is the base line of maximal stability extrapolated from the fluorescence ratio data of β-lactoglobulin in a solution of 0.005M Na₂SO₄ pH2 using the temperature range 25-65°C and extended for the entire temperature range of investigation.

Figure 6a. Effect of cooling from 20°C to -12°C without freezing followed by reheating to 95°C on the fluorescence ratio signal of the extrinsic hydrophobic fluorophore ANS complexed with β-lactoglobulin in 0.1M NaCl pH2. ANS binds to many more sites than there are tryptophans, providing a more global fluorescence unfolding signal.

Figure 6b. Temperature dependence of fluorescence emission intensity (arbitrary units) of retinol-β-lactoglobulin complex at 480 nm. β-lactoglobulin is thought to be a transporter of small hydrophobic molecules and retinol (vitamin A), a fluorophore, is one of the molecules most likely to be this protein's natural substrate in the intestine. This provides another check that the protein is both unfolding near 0°C, and refolding upon further cooling.

Figure 7a. Typical trace of the heat capacity of β-lactoglobulin in solution of 0.1 M NaCl pH2, as monitored by high sensitivity microcalorimetry (left panel). In the right panel rough curve is raw heat capacity data, smooth curve is the estimate of excess heat capacity (according to the classical thermodynamic theory for folding-unfolding transitions of proteins) of the protein undergoing heat denaturation.

Figure 7b. Calorimetric heat capacity changes of β-lactoglobulin in glycine buffer pH2 in a typical heat denaturation experiment compared to a heat denaturation experiment in which the protein was dissolved in a glycine buffer containing 2 M GndHCl. In the left panel curves 1a, b, c represent the heat capacity during the first, second and third consecutive scans of the protein in glycine buffer and curve 2 shows the heat capacity during the first scan in glycine buffer containing 2 M GndHCl. Right panel is as in Fig. 7a.

Figure 7c . shows the effect of increasing glycerol concentrations in 0.1 M NaCl, pH2, on the microcalorimetrically observed heat denaturation of β-lactoglobulin (left panel). Right panel is as in Fig. 7a.

Figure 8. Calorimetric behavior of concentrated

(12% weight/volume) β-lactoglobulin in 0.1 M NaCl, pH2 as measured in a Perkin-Elmer DSC4. The curve beginning at

40°C represents the continues cooling to -160°C. Sample was then rewarmed to -2°C and second curve was recorded from - 2°C to -40°C

Figure 9. Far UV circular dichroic spectra of heat denaturated jβ-lactoglobulin by extreme temperatures and high concentrations of denaturants in comparison with denatured artificial peptide Glu-Lys-Lys-Leu-Glu-Gln-Ala (SEQ ID NO:1). Figure 10a. Dependence of far UV circular dichroic spectra on the thermal history of β-lactoglobulin in 0.1 M NaCl, pH2.

Figure 10b. A modified intrinsic base state spectral analysis (MIBS) of a far UV circular dichroic spectrum of β-lactroglobulin at -0.5°C.

Figure 10c. A MIBS analysis of a far UV circular dichroic spectrum of β-lactoglobulin at 40°C. during rewarming.

Figure 10d. A MIBS analysis of a far UV circular dichroic spectrum of β-lactoglobulin at 65°C during rewarming.

Figure 11. Temperature dependence of the fluorescence ratio of horse skeletal muscle myoglobin (Panel 1, Panel 3) and β-lactoglobulin (Panel 2) as influenced by pH and thermal history.

Figure 12. Temperature dependence of the fluorescence ratio of actin (Panel 1), of human fibronectin (Panel 2), of trypsin (Panel 3) and α- chymotrypsin (Panel 4) as influenced by thermal history.

Figure 13. Temperature dependence of the fluorescence ratio of bovine serum albumin (Panel 1), of lysozyme at pH 4.8 (Panel 2), of lysozyme chitotriose complex at pH 5.5 (Panel 3) and of lysozyme chitotriose complex at pH 7.5 (Panel 4) as influenced by thermal history.

Figure 14. Schematic diagram illustrating the spectrofluormetric apparatus optimizing the measurement of the effects of protein destabilization according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the applicant's discovery that a large proportion of mammalian proteins suffer significant structural destabilization upon cooling to about 0°C, which persists during subsequent rewarming. This discovery has led to design of the presently disclosed method for characterizing the stability of proteins in frozen concentrated systems such as is encountered in the industrial processing of freeze dried pharmaceuticals, foods, and cell cultures. In addition this system can be used to scan cooled or warmed pathogenic host cells exposed to a potential therapeutic agent or stress to determine the extent of protein instability induced in the cells.

The presently disclosed method can be used, for example, to compare healthy cells at 37°C, malignant cells at 37°C and both groups of cells exposed to some stressful temperature such as 0°C, both in the presence of and absence of compounds which could potentiate protein instability preferentially in the cancer cells.

The presently disclosed system will not be limited to analysis at low temperatures such as about 0°C, but the crucial observation made by the applicants that 0°C will greatly potentiate the destabilizing effects which many compounds have on proteins generally, and that this destabilization can persist for days subsequent to reheating means that scanning at low temperature is an important feature.

Specifically, the present invention provides a method of evaluating agents for enhancing the stability of protein formulations comprising:

(a) providing a first sample of a substantially homogeneous protein formulation;

(b) determining a range of temperature at a constant stress over which the protein formulation remains in an acceptable range of stability;

(c) measuring the fluorescence emission spectrum in the range of 300-500 nm of the protein formulation when excited by a light beam in the range of 270- 700 nm to provide a first spectrum, the measuring being performed at a temperature within the range determined in step (b);

(d) providing a second sample of the substantially homogeneous protein formulation wherein the protein is substantially heat denatured; at a constant stress; (e) measuring the fluorescence emission spectrum of the second sample in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a second spectrum, the measuring being performed at the constant stress;

(f) comparing the first and second spectra to determine lambda (1) and lambda (2), such that lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between the first and second spectra;

(g) providing a third sample of the substantially homogeneous protein formulation such that the protein therein is maximally stabilized as evaluated by an acceptable level of activity at the constant stress over as wide a temperature range as possible;

(h) repeatedly recording fluorescence intensity at lambda (1) and fluorescence intensity at lambda (2) and forming a ratio of fluorescence intensity at lambda (1) divided by fluorescence intensity at lambda (2) of the third sample of the protein formulation excited by a light beam in the range of 270-700 nm; simultaneously the temperature of the sample is scanned and sampled, beginning at a reference temperature at the start of the recording process, and terminating at either the maximum or the minimum temperature of the range of stability at the end of the recording process to provide a stable base line;

(i) providing a fourth sample of the substantially homogeneous protein formulation such that the protein formulation has been maximally destabilized over the full temperature range of interest as determined by standard biochemical means such as enzyme activity and such that the destabilization has been achieved by heating the protein formulation to the lowest temperature, T, in the temperature range of the heat denaturation, which can be demonstrated to effect irreversible loss of acceptable activity over the full temperature range of interest wherein activity can be measured, at the constant stress;

(j) repeatedly recording fluorescence intensity at lambda (1) and fluorescence intensity at lambda (2) and forming the ratio of the fluorescence intensity at lambda (1) divided by fluorescence intensity at lambda (2) of the fourth sample of the protein formulation excited by a light beam in the range of 270-700 nm; the temperature being scanned in cooling direction starting from the temperature of irreversible loss of acceptable activity (T) as determined in step (i), at constant stress, the minimum temperature of measurement being determined by the lowest temperature of interest to provide an unstable base line;

(k) providing a fifth sample of the substantially homogeneous protein formulation which is a control sample and is of a composition representative of the useful product which is to be stabilized.;

(1) repeatedly recording fluorescence intensity at lambda (1) and fluorescence intensity at lambda

(2) and forming the ratio of the fluorescence intensity at lambda (1) divided by the fluorescence intensity at lambda (2) of the fifth sample of the protein formulation excited by a light beam in the range of 270-700 nm; simultaneously the temperature is scanned and sampled starting from the reference temperature at the start of the recording process and terminating at either the maximum or the minimum of the temperature range of interest, at the constant stress at the end of the recording process to provide the control ratio curve; (m) providing a sixth sample of the same composition as the fifth sample, but containing an agent to be tested as a stabilizer or destabilizer.

(n) determining a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in the sixth sample while the temperature is simultaneously scanned and sampled starting from the reference temperature and either heating to the maximum of the temperature range of interest followed by recooling to the reference temperature or starting from the reference temperature and cooling. to the minimum of the temperature range of interest followed by reheating to the reference temperature or, optionally, to any temperature in the temperature range of interest, to provide the test ratio curve to estimate the effectiveness of the test agent to stabilize or destabilize, at the constant stress;

(o) evaluating the ability of the agent to enhance stability of the sixth sample protein formulation by comparing the fifth sample (control) and the test plurality of ratios of the sixth sample

(test), such that stability will be increased whenever the difference in the ratio of the unstable base line and the ratio of the test protein formulation is larger than the difference in the ratio of the unstable base line and the ratio of the control protein formulation and both differences are of the same sign for any given temperature; and optionally

(p) evaluating quantitatively the ability of the agent to enhance stability of the sixth sample protein formulation by comparing (1) the value of the control ratio curve (fifth sample) minus the value of the stable base line ratio curve (third sample) divided by the value of the unstable ratio curve (fourth sample) minus the value of the stable base line ratio curve to (2) the value of the test ratio curve (sample 6) minus the value of the stable base line ratio curve divided by the value of the unstable ratio curve minus the value of the stable baseline ratio curve such that the protein will be taken to be stabilized if the value of (2) is less than (1) and the degree of stabilization will be taken as the value of (1) minus (2), at any given temperature.

It will be appreciated that once a stable base line, unstable base line and control experimental ratio curves are determined for a given substantially homogeneous protein preparation, the effect of any agents on the stability or instability of the protein may be determined by the above methods such as those detailed in steps (m) through (o), above. The present invention, therefore, also will be recognized to include a method of evaluating proteins to determine these baseline curves as well as a method to test agents once these curves have been determined.

An acceptable level of protein formulation stability can be determined by any manner known to those skilled in the art. Typically if the preparation possesses an activity which is the property of interest, then destabilization will be a level of activity which falls outside of a defined range of acceptable activities at a reference temperature. The reference temperature can be any temperature that the investigator wish to use as a standard. Typically however, the most useful reference temperature will be the temperature at which the protein formulation is expected to act in its application. A maximally stabilized protein formulation is one to which well known stabilizers such as certain salts, amino acids or polyols have been added in significant quantity, such that an undetectably small proportion of protein molecules is in the unfolded state.

The range of acceptable activity would then be defined as a range from a minimal to a maximal level of activity which meets any established or appropriate guidelines for the use of the material. These guidelines might, for example, be set by regulatory agencies such as the Federal Food and Drug Administration or by the market place in which a preparation with other than the required activity cannot be sold or otherwise used. Specific examples include but are not limited to the following.

If the activity of interest is enzymatic activity, the determination of the constant stress over which the activity is in an acceptable range would be met by exposing a preparation to systematically varied values of the stress, such as pH, over a temperature range both above and below the reference temperature . After each exposure to a test temperature, the enzyme preparation would be returned to the reference temperature and its activity measured. If the activity falls within the levels specified in the guidelines then the test temperature of exposure is within the range of acceptable stress. Measurement of the activity would generally consist of determining the rate at which the enzyme converts substrate molecules to product molecules. This rate would be compared to the rate referred to in the guidelines and if it was within the range specified by the guidelines the rate would be deemed acceptable . The actual determination of the rate of turnover could be any of the methods known to one skilled in the art such as the measurement of the change of the level of the absorption of light at a particular wavelength at which said light is absorbed almost exclusively by either the substrate or product molecules.

If the protein preparation of interest is an antibody preparation, acceptable activity will be determined at the reference temperature after exposure to each level of stress in the range of stress being tested as a level of binding of an antigen which is in the range of levels of binding as referred to in the guidelines. The level of binding can be measured by one of number of standard techniques as, for example, light scattering from immunoprecipitation or the formation of a colored product in an ELISA reaction.

If the protein preparation of interest is a pesticide preparation, then the range of levels of acceptable activity will be defined at the reference temperature, for each value of stress, in the range of stress, by evaluating the protein preparation with a test group of target organisms to demonstrate an amount of lethality in an acceptable range.

If the protein formulation is a vaccine then the range of levels of acceptable activity will be determined by measuring at the reference temperature for each level of stress in a range of stress a rate of induction of protective antibody response in a group of target organisms which rate will be deemed acceptable if it falls within the range defined by the guidelines. In addition a separate maximum level of acceptable activity may be defined such that the level of acceptable activity is a rate of infection of target organisms at the reference temperature after exposure of the vaccine preparation to a test level of stress, or a rate of induction of hypersensitivity in a group of target organisms after exposure of the vaccine preparation to a test level of stress, which is outside the range of rates specified in the guidelines.

If the protein formulation is a hormonal therapy product a range of levels of acceptable activity will be determined at the reference temperature after exposure to each level of stress in the range of stress by demonstrating that the rate of transformation of target tissues by the product is within the range specified in the guideline. Here the transformation is taken to mean that the target tissue returns to a functional state within the physiologically normal range of activities effected by this hormonal function.

The stable base line for each of these illustrative applications is created by adding agents to the protein preparation such that the temperature range over which the preparation can be returned to the reference temperature and subsequently shown to retain activity in the range defined by the guidelines is as wide as possible.The fluorescence ratio is recorded over this range in the presence of the stabilizing agent and this ratio is then taken to represent the fully stabilized preparation. When activity is measured biologically, the stabilizing agent may have to be removed upon return to the reference temperature, depending on the biocompatability of the stabilizing agent.

Whereas the unstable base line can be determined experimentally from temperatures below the glass transition of water at -135°C to temperatures in excess of 100°C, as a general rule the range of experimentally determinable points of the stable base line is much smaller, typically in the range of about 20°C to about 60°C. Thus the stable base line at very high and very low temperatures usually is a mathematical extrapolation of the experimentally measured curve.

Results presented in the detailed description of this invention indicate that the measurement of the degree of destabilization at low temperatures as measured by the fluorescence ratio method will support a close correlation between the proportion of molecules unfolded and the percent of molecules retaining activity. The method of the invention is designed to correlate the fluorescence ratio with desired activity, but at low temperatures, especially in frozen and lyophilized preparations, the fluorescence ratio is most likely to represent the percentage of the protein molecules unfolded and the extent of the activity is not practically measured by standard assays. Nevertheless the preservation of the native conformation of the protein is the best method of insuring that when the protein formulation is returned to the reference temperature it will be within the range of acceptable activity.

Application of an amount of stress in the increasing direction means increasing the amount of stress, such as decreasing the temperature of the cell environment or increasing the temperature of the cell environment from an optimal or cell culture temperature. Applying an amount of stress in the decreasing direction means returning the cellular environment to an optimal or cell culture condition, such as returning the cell temperature from a temperature above 37°C to 37°C.

Determining the range of stress over which cells are viable is that range of decreasing or increasing temperature from an optimal or normal temperature for a given cell type over which the cell remains viable. Generally, cellular proteins will be substantially denatured at temperatures of about 0°C and greater than about 55°. The agent added in the method of the invention may be any chemical or physical effector which establishes cellular proteins such as, for example, decreasing or increasing pH, radiation (such as, radiofrequency, microwave, ultrasound) and chemical agents, which might include cytotoxic antibodies, cisplaten, cyclosporin, melphalin, nitroureas, thio-TEPA, actinomycin D, bleomycin, doxorubicin, amphotericin B, Cysteamine (with or without other SH compounds), ethanol, lidocaine (and other local anesthetics), polyamines, misonidazoles, 5- fluorouracil, methotrexate, vinblastine and vincristine.

Cell viability can be measured by any manner known to those skilled in the art. If cell instability is to be produced by destabilizing proteins, cell viability will, preferably, be measured by a method which, at a minimum, determines the inability of cells to produce proteins. If cell instability is to be produced by nucleic acid destabilization, the range over which cell instability is produced when subjected to an applied stress will be, preferably, measured by a method which, at a minimum, determines the inability of cells to replicate nucleic acids, such as DNA.

One method of measuring cell viability which may be used in the present invention entails evaluating the integrity of the membrane integrity with a fluorescent dye, such as by the fluorescein diacetate/ethidium bromide assay, as described by Martel et al (Vox Sang 27, 13-20, 1974). In this assay, viable cells show a green fluorescent cytoplasm and dead cells show a red fluorescent nuclei under epifluorescent microscopy. Other alternative methods can be found in Atkinson et al (Biochemical Engineering and Biotechnology Handbook (1991) Second Edition, Stockton Press, NY, NY) and references cited therein.

Cell survival or viability can also be evaluated by a number of biochemical functions including DNA, RNA and protein synthesis, respiration and glycolysis. For evaluation of cancer cells, one of the most important properties of the cells is their reproductive integrity, as measured by colony-forming assay (cell survival), which relates directly to the ability of a cancer to maintain growth. Such assays are generally described in Tannock and Hill (The Basic Science of Oncology (Eds.) Pergamom Books, Inc., NY, NY (1987)).

Although it is expected that the presently disclosed method will be applicable to cell suspensions which are substantially homogeneous in cell type, it is also likely that the present method will be useful in suspensions of mixed cell populations. Substantially homogeneous, as used herein, refers to a population of cells where at least about 75% of the cells are of one type, preferably at least about 85%, more preferably greater than 90%.

The presently disclosed invention further provides a spectrofluorimeter system for in vitro assessment of cell cultures or frozen or lyophilized protein formulations which combines the techniques of laser scanning microscopy (LSM) with a double photon process fluorescence measurement coupled to direct fluorescence spectrum recording and subsequent determination of specific fluorescence ratios. The system of the present invention includes, preferably, a pumped, frequency doubled dye-laser tuned to a wavelength in the range 500-700 nm aimed at a lens system capable of pivoting so as to scan the sample in the X-Y plane. The beam of exciting light is passed through the lens system and focussed on the plane of a collection of cells, such as a suspension of cells or a colony of cells growing on a solid surface, more particularly, a culture of either malignant or normal (cells) or alternatively, a frozen or freeze-dried protein formulation. The light beam path can be systematically tilted so a scan is created. The back scattered fluorescent light is collected at a short wavelength and a wavelength to be determined experimentally, is exemplified below, and used to form the intensity ratio.

Laser scanning microscopy (LSM) is a technique which has been recently developed by two separate groups (J.F.White, J.F., Amos,W.B. and Fordham,M. J.Cell.Biol. 105 (1987) 41-48; V.Wilke. Scanning 7 (1985) 88-96). This technique has been applied to confocal microscopy to restrict sample illumination to a region much smaller than the size of a single cell, increasing the sensitivity and the precision of the observation. The use of the laser allows a point by point scan with an illumination sphere which is less than 10 microns radius. The ability to accomplish this is dependent upon the characteristics of the laser source. This technique allows fluorescent studies of very localized phenomenon in situ in cells as reported by other authors (Paddock, S .W. J.Cell Science 93 (1989) 143-146; Summers, R.G, Strieker, S .A. and Cameron,R.A. Methods Cell Biol. 38 (1993) 265-287; Piston, D.W., Sandison,D.R. and Webb,W.W. Proc.SPIE 1640 (1992) 379-390; Guild, J.B. and Webb,W.W. Biophys.J. 68 (1994) A290).

Recently, this technique of LSM has been augmented by a less destructive method of in situ investigation of living cells. Instead of using a direct UV incident beam to activate the chromophore sites, the absorption of two successive but almost coherent incident photons on the chromophores is made possible by the use of lasers. This technique is called the two-photon absorption technique. The potential to use a two-photon absorption process has been developed by Webb and co-workers (Piston, D.W., Sandison,D.R. and Webb,W.W. Proc.SPIE 1640 (1992) 379-390; Guild, J.B. and Webb,W.W. Biophys. J. 68 (1994) A290; Denk,W., Strickler, J.H. and Webb,W.W. Science 248 (1990) 73-76; Williams, R.M., Piston,D.W. and Webb,W.W. FASEB J. 8 (1994) 804-813).

A presently disclosed application of this technique will consist of a tunable laser with sufficient power output (around 100 milliwatt) to be able to allow the absorption of the double incident photons by the chromophore molecules. The technique of double photon absorption has been shown to be less deleterious for cells than the commonly used fluorescence technique using a single photon absorption (Denk,W., Strickler, J.H. and Webb,W.W. Science 248 (1990) 73-76; Williams,R.M., Piston,D.W. and Webb,W.W. FASEB J. 8 (1994) 804-813). The choice of double photon visible light absorption inevitably entails the loss of photon absorption efficiency as compared to single photon UV excitation. Nevertheless, the double photon technique provides several important advantages over single photon UV absorption, detailed as follows.

(1) Visible light has the ability to penetrate deeply into frozen protein formulation samples or frozen biological samples or freeze-dried protein formulation samples, or freeze-dried biological samples, all of which are opaque in the ultraviolet;

(2) Because in the double photon technique, the fluorescence emission signal is in the UV but the excitation is in the visible, it is straightforward to filter out the scattered excitation light with virtually no loss of fluorescence signal. This is not true in the case of single photon UV excitation where the shorter wavelength of fluorescence emission is very close in wavelength to the excitation signal, and it is difficult to eliminate the excitation signal with high efficiency from the fluorescence signal.

(3) Visible light is non-ionizing; thus, extended or repeated measurement of samples over many hours or even days will not degrade the sample. This degradation is a serious limitation to the standard single UV photon absorption technique.

(4) The efficiency of signal production in the double photon technique is proportional to the square of the power of the incident light. Therefore, since the beam power decreases as 1/ (distance from the focal sphere)², the double photon induced fluorescence emission decreases as 1/ (distance from the focal sphere)⁴. In contrast, the ultraviolet induced emission decreases as 1/distance² from the focal sphere. Furthermore, the cross-section for absorption is much larger for UV so the signal from the unfocussed double photon system will become negligible at a distance much closer to the focal sphere than is the case for the single photon UV system.

Characterization of protein stability results from the capacity to measure specific fluorescence ratio signals at fixed wavelengths chosen to optimize the sensitivity to protein unfolding for the considered protein set. This means, in this embodiment, that the wavelength of the laser must be greater than 500 nm to ensure that the two photon absorption is equivalent to absorption of a single UV photon in the range of 250-300 nm. Therefore, the system consists of a tunable laser such as a copper laser. Copper lasers have been used extensively in spectroscopy to generate tunable narrowband light by pumping a dye-laser (G.A.Naylor, Koprinkov, I.G., Sitja,G., Ring,H and Pique, J. P. J.de Physique IV 4 (1994) 759-762). The same technique can be used with an ND:YAG-pumped dye-laser (Oki,Y., Izuha,T., Maeda,M. Honda, C, Hasegawa,Y. Futami,H., Izumi,J. and Matsuda,K. Japan. J.Appl. Phys. II. Lett. 30 (1990) 1744- 1746). However, the choice of the laser will be determined by the need for a stable frequency of pulse emission of the laser. In general, the technique will use a source with either an ND:YAG or copper gas pumped dye- laser with a spectrum for the dye-laser which can reach emission wavelengths between 500 and 1000 nm. The power range of these dye-lasers is within the range of .1 to 1 watt (Duarte,F.J. and Hillman,L.W. Dye Laser Principles with applications. Acad. Press., New York, 1990 ) with a line width of the order of several femtoseconds and with a pulse frequency of 10 kHz to 50 MHz. In general, the efficiency of double process is proportional to the square of the incident beam power, and to the pulse frequency. Thus, for the investigation of steady state fluorescence ratio behavior over extended time periods, a high pulse frequency and maximum power density is preferred. The presently disclosed system includes a calibrator allowing accurate tuning of the wavelength of the dye-laser. This calibrator has recently been developed by Oki et al (Duckworth,A., Adrian, R.S. and Tozer,B.A. Optics and Laser Technol. 24 (1992) 39-43). This system allows the calibration of the lasers at wavelengths between 220 and 740 nm using an optogalvanic technique (Duckworth,A., Adrian, R.S. and Tozer,B.A. Optics and Laser Technol. 24 (1992) 39-43). This coupling system allows the control of the pulse rate and of the peak shape of the signal.

This embodiment of the present invention preferably includes the capability to focus the laser generated excitation beam to a 10 micron sphere so as to be able to distinguish the fluorescence ratio signal of single cells from the surrounding medium, or to distinguish individual small concentrated particles of protein formulations in frozen or lyophilized preparations from the inert supporting medium. These constraints have been analyzed in the literature (Guild, J.B. and Webb,W.W. Biophys. J. 68 (1994) A290; Denk,W., Strickler, J.H. and Webb,W.W. Science 248 (1990) 73-76; Williams, R.M., Piston, D.W. and Webb,W.W. FASEB J. 8 (1994) 804-813).

Further details of this embodiment of the present invention are in of Figure 14 are described in the following.

Component 1 is a titanium-sapphire pumped dye- laser or copper vapor pumped dye-laser, or neodymium:YAG pumped dye-laser, with an appropriate choice of dye to obtain an excitation wavelength spectrum between 500 and 1000 nm. The choice will be made on the basis of the ability to achieve an emission power between 0.1 and 1 watt with a pulse rate of 10 kHz to 50 MHz with a time rate of the pulse of 1 to 100 femtoseconds. These characteristics allow the definition of optimal experimental conditions for the energy of the beam, i.e., the ability to focus the beam to a diameter of the order of 10 microns and the ability to scan the sample in real time. Component 1 will preferably include a set of controls for the determination of the wavelength with the laser through the use of an optogalvanic wavelength calibrator, and of the pulse size and pulse energy and of the pulse frequency of component 1. The excitation signal is directed to component 2, an XY-scanner device for directing the beam. The excitation signal is then sent from component 2 through component 3, a defocussing lens having a certain aperture and a dispersion for the excitation signal such that ultimate focussing of the excitation signal to a sphere of approximately 10 microns is facilitated.

The excitation signal from component 3 is then sent to component 4, a collimating lens to produce a widened parallel excitation beam. The excitation beam from component 4 is sent through component 5, a semi- mirror, to component 6, an objective lens which condenses the excitation beam and focusses it on a sample, component 7.

Component 7 is exemplified by a culture cell on a plate or in solution, a section of animal or plant tissue or a protein formulation on some translucent substrate. The sample, component 7, is bracketed and held by component 8, a cryostage for maintaining control temperatures. The temperature is measured on the cryostage and is transmitted to component 16, a computer for use in a computation which will generate an output signal to change the temperature on the cryostage according to a predetermined program in component 16.

Optionally, the cryostage, component 8, can be replaced by a more sophisticated XY cryostage with position control allowing displacement of the sample, component 7, in the XY direction of approximately .1 micron per step. This would alleviate the need to use component 2 to change the position of the beam.

Component 9 is an adjustable stage holding component 8. Component 9 adjusts the position of component 8 and component 7, in the direction perpendicular to the incoming excitation beam so as to achieve proper focus of the beam on the sample component 7. If the sample allows the passage of excitation light then the excitation signal passes through component 7, the sample, to the condensing lens, component 10, which focusses the excitation signal on the photo-multiplier tube component 11. Component 11, the photomultiplier tube, detects this forward scattered light from the excitation signal beam, and sends a proportional electronic signal to the computer, component 16, which stores it for use in the fluorescence ratio analysis.

Component 12 is a liquid nitrogen tank which is connected through a control valve allowing the flow of nitrogen coolant to the cryostage, component 8, and the flow is controlled by component 16, the computer, based on the signal that component 8 sends to component 16 which registers the temperature and the program in the computer which directs the change in temperature with time. The component 13 is an optional bandpass filter 300-500 nm which eliminates significant backscatter of incident excitation light, but passes the backscattered fluorescent signal from the sample, component 7, reflected by semi- mirror component 5 to be used when the sample allows passage of excitation signal to the condensing lens component 10.

After the fluorescent signal passes through component 13, the filter, it is focussed on component 15, a second PMT, by component 14, a lens. Component 15 is a PMT which is a Fourier PMT which collects the incident fluorescent signal. Component 15 is able to distinguish between the various wavelengths of light which are incoming and integrates the intensity of fluorescent signal over preset channels of typical width 1 nm for the range 300-500 nm and transmits each integrated value to the computer component 16 which then uses the values to create a ratio file for the generation of fluorescent ratio curves. If the sample, component 7, is opaque to the excitation light such that component 13 is removed, then the integrated signal from component 15 for the wavelengths of excitation light are sent to the computer, component 16, and stored as the backscattered excitation signal.

The set of components 14 and 15 may be replaced by another set consisting of a beam splitter 15a which reflects the incident beam to two beams separately divided towards two micrometers 15b and 15c which in turn direct the fluorescent signals impingent upon them and towards two independent PMTs 15d and 15e.

This embodiment of the present invention can be used in several modes depending on the type and characteristics. One geometric scan will be enough to collect sufficient data concerning the sample using the scanner component 2 or the automated positioner component 8 of the sample. As described above, various samples will be scanned for the determination of the spectrum of the fluorescent signal in the native and denatured states of the proteins. This will allow the determination of the optimal ratio at two wavelengths between 300 and 500 nm to be used to create ratio curves. The calibration will be done on a control sample of cells and culture, or, alternatively, on a protein preparation of interest. The necessary acquisition time for ratio data is expected to be no more than about 1 microsecond per cell as per the calculations of Denke et al, Science, vol. 248, 1990. The XY scanner of component 8 or the beam scanner component 2 will be systematically varied in position at an appropriate rate, and the signal of both backscattered fluorescence and the forward scattered excitation beam or backscattered fluorescence and backscattered excitation beam will be monitored by the appropriate photomultiplier tubes. After the determination of the optimal fluorescence ratio from the calibrated spectra for the determination of the fully native and fully denatured states of the sample, the computer may correlate the fluorescence signal with the scattered signal to avoid artefacts of measurement. Both signals will be intrinsically synchronized. Correlation of the forward scattering excitation or the backscattered excitation signal and the backscattered fluorescence signal will be accomplished by the computer component 16 such that the cells will be accurately identified as having a high fluorescent signal, and a low forward scattering signal or a high fluorescent signal and a higher or lower backscattered excitation signal depending on the relative reflectivity of the medium with respect to the cells.

As one of ordinary skill will appreciate, there have been recent advances in dye-laser technology. As stressed by Webb and co-workers, the use of a relatively high-powered dye-laser has made it possible to focus the laser beam to a 10 μm region to increase the probability of double photon absorption to one per chromophore per laser excitation pulse.

In some cases, the use of a Fourier photomultiplier is advantageous over the use of two separate photomultipliers synchronized for the collection of data and coupled to two independent monochrometers that are mechanically driven, because analysis of incident spectra from individual calls or microregions of a protein formulation from the Fourier photomultiplier will be possible after the experiment. On the other hand, the advantage of two independent photomultiplier tubes coupled with their respective monochrometers is rapidity of measurement as soon as both frequencies for the determination of the fluorescence ratio are known. Indeed, the acquisition of these ratios will be quicker and more efficient; but the information at other wavelengths will not be available. The particular experimenter will have to decide which is the most efficient set-up for their particular needs.

The determination of an optimal fluorescence ratio is based on two criteria: (a) the intensity at the wavelength used in the denominator should show as close to zero response as possible to massive denaturation of the whole protein content; and (2) the intensity at the wavelength used for the numerator should show maximal sensitivity to the same treatment.

Experience with a series of model proteins indicates that these wavelengths will probably be close to 320nm and 350nm respectively. Thus, the intensity is measured at these wavelengths using a photomultiplier tube and the ratio of the fluorescence intensity: e.g., l₃₅₀/I₃₂₀, is calculated. A fraction of the forward scattered light is also sent to and collected by a separate photo multiplier tube. This signal indicates whether the beam is focussed on a region of locally high protein content, such as a cell, and thus allows separate analysis of regions of variable protein content such as the intracellular vs extracellular space. The apparatus of the present invention, therefore, will be capable of providing an estimate of the fraction of unfolded protein at each temperature of measurement individually and separately for each cell in a culture of cells allows one to correlate the response of cells to the externally applied stress with the fluorescence ratio signal of the cells. In addition, the apparatus will be able to provide detailed or local ratio information, as opposed to global information, which may be correlated to the chemical or thermal history of a sample.

As applied to tumor therapy, control runs will consist of fluorescent ratio measurements on cultures of healthy non-malignant cells and other analogous measurements on malignant cells over the full temperature range for which it has been determined that these cells can grow and be maintained in culture. These fluorescent ratio measurements will be extrapolated into temperature ranges of interest in which the cells are known to suffer some injury. The measurements thus provide the "uninjured" baseline defining the maximal stability of the cellular proteins of each group in vivo.

A second group of control cells will be heat killed near 100°C or, alternatively, by strong denaturants such as urea, guanidine hydrochloride (GndHCl), extreme pH, etc. Fluorescent ratio measurements of these killed cells will be taken over the full range of temperatures of interest. They represent the baseline of "fully" unfolded cellular proteins. This is generally a higher ratio throughout the temperature range. Measurements taken on samples of interest will show a fluorescence ratio somewhere in between the two baselines, but significantly closer to the uninjured baseline unless the cells are killed or are significantly injured.

The magnitude of the ratio signals from the stressed cancer cells will then define a quantitative measure of the forced denaturation of their proteins. This provides a basis for an empirical approach for evaluating compounds that, for example, are preferentially taken up by the malignant cells, either at 37°C or lower temperatures, and which subsequent to uptake exacerbate the general denaturation of cellular proteins near 0°C and/or lead to increased persistence of abnormal folding of cellular proteins upon return to physiological conditions. One of ordinary skill will appreciate that the presently disclosed method is useful to evaluate compounds other than those which are preferentially taken up by the cells, but rather should also be useful to evaluate, in general, the effects of external agents or processes on protein stability. Use of this method will lead to accumulation of data on various cell types at low temperature correlating survival after rewarming with the extent of denaturation near 0°C as indicated by the fluorescence ratio and/or the extent of refolding hysteresis indicated by fluorescence ratio analysis upon rewarming. These effects are generally associated with increased tissue mortality. The method will allow quantification of both the magnitude and kinetics of this process.

The presently disclosed invention will also be useful in evaluating or monitoring the effect of preservation methods on the stability of proteins whether in solution or in cellular preparations. That is, the method will be useful, for example, in evaluating methods as well as quality control or quality assurance of protein or cellular preparations. The present invention is based on a new thermodynamic model of protein folding-unfolding based on the assumption that the physics of protein unfolding is a complex process involving the breaking of various types of internal bonds, increase in configurational entropy of the whole protein polymer, and the free energy of solvation of newly exposed residues. The present inventors have, in addition, separated the free energy into three fundamental categories, each with its own associated mathematics to more completely describe the process of protein dynamics.

In the first category are combined the internal bond breaking and configurational entropy changes and the form is assumed to be the same as that of the classical theory, that is, ΔC_p is constant for this process and the free energy is characterized by the sum of a linear and a linear times logarithmic temperature term, i.e. A*T+B*T*log(T). The second category is solvation of buried groups. This process is assumed to have a linear temperature dependence of ΔC_p, which translates to additional quadratic temperature terms contributing to the free energy, i.e. C*T². The third category are interfacial tension terms which are associated with newly exposed hydrophobic groups which fail to become solvated. These terms have an Eötvös form for interfacial free energy (Adamson, A. In: Physical Chemistry of Surfaces, 49, 1982), i.e., a constant minus temperature divided by a reference temperature all raised to a rational power that empirically is observed to range from about 0.5 to 1, (D- (T/T_c))^m.

In response to this postulated variation in the free energy of solvation, the applicants generally refer to this approach as the differential solvation (DS) theory.

Analyzing the fluorimetrically derived free energy data using this approach leads to equations for heat capacity, enthalpy and Gibbs free energy which predict an array of previously undiscovered behaviors, including: (1) some globular proteins probably do not fully cold denature at any subzero temperature when cooled in the absence of strong denaturants; (2) the ΔC_p of cold denaturation can become negative at temperatures well below the temperature of heat denaturation; (3) the denaturational enthalpy can reverse sign and become endothermic, as it is at high temperature; (4) at still lower temperatures, in the case of proteins which do cold denature, ΔG_den may reverse sign to become positive again thus causing an ultra-low temperature restabilization of the native state.

Furthermore, detailed analysis of the free energy curve for β-lactoglobulin, a small protein from whey believed to be a transporter of hydrophobic molecules in the mammalian intestine which has been used herein for illustrative purposes, suggests that in the neighborhood of 0°C several order transitions occur in the hydrophobic residues which add significant stabilization free energy to the molecule between 0°C and -5°C. This is due, in part, to an increase in the Eötvös interfacial free energy terms. The circular dichroic (CD) analysis referred to as modified intrinsic base state (MIBS) spectral analysis leads to the discovery that this packing transition is conversion of β-sheet and random coil to α-helix and β-turn. Furthermore, the presently disclosed use of fluorescent ratio analysis on supercooled or frozen solutions of randomly chosen water soluble proteins of widely varying amino acid composition, biological function, and molecular weight shows a remarkable similarity of structure in the resulting curves, leading inescapably to the conclusion that there is, at a minimum, a general destabilization of water soluble proteins near 0°C.

As further exemplified below, application of the presently disclosed differential solvation theory allows for the measurement and analysis of structural changes of polymers. Specifically, as further detailed below, the present invention provides methods and apparatus for evaluating agents, such as chemotherapeutic agents which cooperatively or selectively destabilize biological polymers, such as proteins. Alternatively, the present method allows optimization of additives and temperature ranges to stabilize proteins during storage and formulation of pharmaceuticals at low temperature by allowing monitoring of the real time stability of proteins. In addition, the present method allows discovery of optimal temperature ranges and cryoprotective additives for the stabilization of proteins in cells and tissues cooled to below 0°C during cryopreservation.

The invention will be further described by way of the following non-limiting examples which are representative of the invention.

EXAMPLES

The following procedures and materials are general to the examples disclosed below. Chemicals and Solutions:

Bovine β-lactoglobulin type A (BLG) was purchased from Sigma Chemical Company and used without further purification. The protein was found to be homogeneous by both SDS-PAGE electrophoresis using 8-25% gradient or 20% homogenous gels (Pharmacia Phast system), and N-terminal sequencing which was done on a Hewlett Packard model G1000S sequenator. Ultra-pure urea and GndHCl (Guanidine hydrochloride) were obtained from GibcoBRL (Gaithersburg, MD) and USB, retinol from Sigma (St. Louis, MO), and 8-anilino-1-naphtalenesulfonate magnesium salt (ANS) from Eastman (Rochester, NY).

Urea was additionally purified according to the method of Prackash et al (Arch. Biochem. Biophys. (1981) 219: 455). The working concentrations of both denaturants were confirmed by densitometry or refractometry (Kawahara, K. and Tanford, C. J. Biol. Chem. 241,13,3228, 1966; Nozaki, Y. Methods in Enzymology, XXVI, 43, 1972).

All measurements in this work were done in 0.1M NaCl pH2 or 0.05M Glycine buffer with varying pH as indicated unless otherwise noted.

Retinol and ANS concentrations were measured spectrophotometrically using ε₃₂₅=46000 and ε₃₅₀=6240, respectively.

Retinol was added to the protein in a 1:1 molar ratio as an ethanol solution so the final content of ethanol never exceeded 2%.

Fluorescence:

The cold and heat denaturation of BLG were detected using an SLM-800C computer controlled fluorometer monitoring the intensity of emission fluorescence intensity over the wavelength range 320-350nm. The emission monochromator was set to step 1 or 5nm with excitation at 280nm. The excitation wavelength is set to 280 nm because that is near the maximum of absorbance of both tryptophan and tyrosine. The emission range 320 to 350 nm is known to include the optimum wavelengths for ratio measurement of these β-lactoglobulin solutions. Measurements were made continuously and averaged over two seconds at each emission wavelength in the forward direction, with reset to 320nm taking two seconds. Heating or cooling scans were conducted between -35°C and 100°C at a rate of 0.1 to 0.5°C/min using a computer controlled circulating bath (Neslab, Portsmouth, NH).

Based on the fluorescence the intensity ratio at 350nm to that at 320, 325, 330nm, and so on, was calculated. A 1cm x 0.2 cm cuvette was filled with the appropriate protein solution at 0.08-0.1 mg/ml. Protein concentration was determined from absorbance at 278nm using coefficient E^1cm'^1%=9.6 (Byler et al. Byopolimers,22,2507, 1983).

Chemically induced cold and heat denaturation of β-lactoglobulin, was carried out using urea (0-10M) or GdnHCl(2-8.1M) (0-8.2M) in 0.1M NaCl at a pH2.

Fluorescence Ratio Method:

The most rigorous assessment of the degree of unfolding of a globular protein in dilute solution as measured by fluorescence utilizes the extent of fluorescence intensity change at fixed wavelength. Unfortunately, practical considerations preclude its easy use. Slight changes in the position of filters and cuvettes as well as protein concentration from run to run cause deviations in absolute fluorescence intensity of up to 100%, making the establishment of accurate baselines very difficult. To overcome this difficulty the ratio of fluorescence intensity at two different wavelengths can be used, effectively normalizing the measurement. This introduces the serious additional problem that the fraction of unfolded protein may not be a linear function of the ratio. To overcome these limitations, a number of relationships were developed which allow testing of the extent of deviation from linearity.

The general linear relationship of fluorescence intensity to fraction unfolded at fixed wavelength, λ₁ , is:

where F_u = fraction protein unfolded, Λ₀ = absolute fluorescence intensity of the folded protein, Λ₁ = absolute fluorescence intensity of the fully unfolded protein, and Λ = measured fluorescence intensity of a mixture of the folded and unfolded forms. Thus, the ratio of measured fluorescence intensity at λ₁ to that at λ₂ is:

If linearity is assumed, this relation simplifies to:

Rearranging these allows development of the following expressions for the exact fraction unfolded and the apparent fraction unfolded:

where r is the ratio:

The ratio of the

F to the

will deviate most from 1 at

This ratio of the two unfolded fraction parameters, where

and is

given by:

the deviation of this expression is proportional to the deviation from linearity. Although this test is valid only for the particular protein and the wavelengths employed, one of ordinary skill in the art will appreciate it has a straightforward physical interpretation. The best wavelength for the numerator is that which shows the maximal change in fluorescence intensity in going from the folded to the unfolded state of the protein. The optimal wavelength for the denominator is one displaying as close to constant luminescence as possible.

An isothermal titration with urea over the temperature range 10°C to 50°C was used to measure luminescence values for fully unfolded protein, allowing the calculation of the deviation ratio at

The ratio of maximum deviation, using Intensity _{350 nm}/Intensity_{330 nm} was always between .95 and 1.05.

One of ordinary skill will appreciate that the fluorescence ratio method has been used in isothermal titration experiments with denaturants such as urea and GdnHCl. In such experiments the proportion of unfolded protein is determined by subtracting the initial ratio in denaturant-free solution from the final "fully unfolded" ratio at the highest possible denaturant concentration, then using that as a normalizing factor for the measured ratio values at intermediate concentrations of denaturant.

This methodology can be problematic for several reasons. Each set of points extrapolates to just one estimate of fraction unfolded at zero denaturant concentration for each temperature examined. The necessity of making the extrapolation means introducing an additional set of assumptions about the dependence of the free energy of unfolding as a function of denaturant concentration, and there are several different models for this in the literature (Wyman, Jr.Adv. Prot. Chem. 19, 223,1964; Greene, Jr. and Peace, C.J. Biol. Chem. 249,5388, 1974; Aune, K.,Tanford, C. Biochemistry, 8, 4586, 1969; Tanford, C. J. Am.Chem.Soc. 86, 2050, 1964; Tanford, C. Adv. Protein Chem. 24, 1, 1970). Even if the model for extrapolation is reasonably accurate, it is only valid if the structural changes induced are the same subset induced by temperature extremes in the absence of denaturant. Thus, if there is more than one two-state transition, separated on the temperature scale, then the denaturant may "mix" them at intermediate temperatures. Finally, most standard denaturants preclude CD measurements in the valuable region below 215 nm.

As a result of these concerns a different method of measurement is central to the presently disclosed method. The denaturant concentration is held constant, and temperature is scanned very slowly (0.1-0.5°C/min), while the fluorescence ratio is monitored several times per minute. This method requires that baseline behavior be determined for folded and fully unfolded species. For the fully folded baseline the ratio in 5 mM Na₂SO₄, pH 2 was used because Na₂SO₄ is demonstrably stabilizing at high temperature. Since the baseline so obtained is virtually flat from +25°C to about +65°C, it can be extrapolated with considerable confidence to temperatures as low as -20°C and as high as 100°C.

The determination of the fully unfolded baseline is considerably more problematic. Ideally, one would like to find a treatment which fully unfolds the protein reversibly over a wide temperature range, such that the unfolded state has essentially the same free energy function as the unfolded protein in dilute electrolyte over the same temperature range. Standard denaturants such as urea and guanidine hydrochloride do not meet these criteria. They work by binding to numerous sites interior to the native state and thus add a significant negative enthalpy of solvation to the free energy of the unfolded state. Thus, some groups which have a significantly positive free energy of solvation in dilute electrolyte, and would stay partially buried in the protein as it is unfolded by temperature alone, would experience a negative free energy of solvation in concentrated denaturant, leading to a more unfolded state.

A second related problem is the nature of water itself. Water binds to many sites on the protein and in the aggregate makes a dominant contribution to the total free energy of unfolding. A multimolar concentration of highly polar denaturant fundamentally alters the thermodynamics of water (Oguni, M. and Angell, C. J.Chem.Phys., 73, 4,1948, 1980) and thus of the equations describing transfer of bulk water to the protein interface. The CD spectrum of the protein at high temperature has been examined with and without concentrated denaturant. The ellipticity between 215 nm and 250nm has been determined to become more negative (as compared to the native state) with thermal unfolding in dilute electrolyte, but more positive when concentrated denaturant is also present at high temperatures (Ananthanarayanan and Ahmad, Can J. Biochem., 1977). Thus, the limiting structure is different when denaturant is absent.

To deal with these difficulties, the recorded ratio of protein has been used, irreversibly heat denatured in 0.1 M NaCl at 102.5°C, from -15°C to 100°C. When heating to 102.5°C was repeated the fluorescent ratio returned to the same value above 90°C. In 0.1 M glycine buffer the heat capacity also returned to the same value during the first reheating scan. Nevertheless, the heat capacity above 90°C increased significantly after the first exposure to high temperature in 0.1 M NaCl. This is probably due to some precipitation induced by the NaCl at high temperatures, but it might mean that whatever permanent change was induced by exposure to 102.5°C affected global structure sufficiently to alter the heat capacity. The constancy of the ratio signal above 90°C indicates that the degree of exposure of the tryptophans to the solution is the same, even after permanent heat denaturation.

Thus, the fluorescent ratio signal indicates that the signal from protein unfolded permanently by heat denaturation at 102.5°C, and the fluorescent ratio signal of protein reversibly unfolded will be nearly the same over the whole temperature range in which measurements have been taken. Furthermore, using an actual experimental curve allows one to account for temperature variation of the ratio signal, whereas extrapolation assumes a temperature independence.

To lend further support to the present approach the upper baselines were calculated by linear extrapolation of the constant ratio signal above 90°C. This yielded slight shifts in the position of the free energy curve at low temperatures, but no qualitative change in the predictions of the physical behavior of the protein. A representative sample of the raw data is shown in the inset to Figure 1.

Once all the precautions for detecting protein unfolding by fluorescence are taken and the proper base lines are obtained, as described in (Eftnik, M. Biophys. J. 66,482, 1944), the fluorescence ratio data can be transformed to a fraction protein unfolded (f_u) and an apparent mass action constant K calculated using the relationship K=f_u/(1-f_u) and a free energy of unfolding ΔG_u=-RTlog_e (K) may be calculated as a function of temperature based on the two state folding model (Pace, Critical Reviews in Biochemistry (1975); Santoro, Biochemistry, (1988) 27:8063; Kellis, Biochemistry (1989) 28:4914, Pace (1990) TIBTECH).

In order to corroborate detection of cold denaturation by the intrinsic fluorescence of β-lactoglobulin, the fluorescence of the natural transport substrate of the protein was measured as the fluorescence intensity of the retinol-BLG complex, excited at 342nm, and using emission intensity at 480nm (Fugate BBA, 625 28- 42 1980). ANS bound to BLG was also used (excitation 350 nm, emission 480nm) with a final concentration of 100 μM. This chromophore is known to bind to the protein at numerous hydrophobic sites located in the interior of folded protein, displaying an increased fluorescent intensity as a result. The relatively non-specific binding makes this a potentially more sensitive test of global unfolding than intrinsic fluorescence. Circular Dichroism (CD) Spectroscopy:

CD spectra in the far UV region (260-185 nm) were recorded at stepped constant temperatures in the range of from -15 ° C to 100 ° C using either a J500C or a J710 spectropolarimeter (JASCO, Easton, MD). Experiments were conducted using jacketed cells with 0.1 or 0.05 cm path length and protein concentrations 0.2-0.4 mg/ml. The results are presented in terms of residual molar ellipticity [Θ] deg² cm² dmol^-1 (molecular weight 18365 D, 162 residues). In order to analyze the experimental curves, several computer deconvolution programs were attempted (CCA, Lincomb, VARSELEC, SELCON, and a proprietary method disclosed in copending U.S. application Serial Number 08/409,064, filed March 23, 1995 and several basis states including basis states from Lincomb). The deconvolutions by the Lincomb program (Perczel, A. et al. Anal. Biochem. 203,83, 1992) and the code disclosed in the copending application utilize five basis vectors over the range 195 nm to 240 nm in 1 nm steps associated with α helix, β sheet, β turn, aromatic residues and disulfide bridges, and random coil and appeared to provide results most consistent with X-Ray data.

Differential Scanning Calorimetry:

High sensitivity differential scanning calorimetry was performed using DASM 1, DAMS 4 and DASM 4M calorimeters at a heating rate of 1°C/min and protein concentration 0.4-2 mg/ml. Before each experiment the protein was extensively dialyzed against the control buffer and all experimental curves corrected for an instrumental base line obtained by heating the dialyzing buffer. Thermodynamic analysis of the excess heat capacity function obtained in these experiments was applied according to the method of Filimonov et al, (1982) Filimonov, V.V., Potekhin, S.A., Matveev, S.V. and Privalov, P.L. (1982) Thermodynamic analysis of scanning microcalorimetric data. Mol. Biol. (U.S.S.R.), 16, 435- 444. The thermal behavior of BLG in the temperature region +40°C to -160°C at a heating respectively cooling rate of 5°C /min was studied with Perkin-Elmer DSC-2 calorimeter also. The protein concentration was 120 mg/ml and the sample volume was 15 μl.

EXAMPLE 1

Fluorescence Studies

Figure 1b displays free energy data for β-lactoglobulin in dilute NaCl and a best-fit least squares curve, according to the classical constant heat capacity theory, for the high temperature data. Using no data whatsoever from calorimetry to create the curves shown, it can be seen that the calorimetrically measured enthalpy and heat capacity at high temperature are predicted quite well by a classical least squares fit. In contrast, there is a complete inability to predict the low temperature free energy data. In addition, Figure 8 shows a complete failure to observe any exotherm in the calorimeter in the range +40°C to -160°C. These results indicate that the classical theory is seriously in error at low temperatures. Moreover, this shows that throughout the range over which the fluorimetric and calorimetric data are available they are consistent, supporting the contention that the fluorimetric data accurately reflect the proportion of unfolded protein.

In Figure lb, the best fluorimetric free energy data has also been fit over the entire temperature range by an equation containing terms for solvation and interfacial energy. That equation has the following form:

ΔG=A+B* T+C* T*Ln (T) +D* T² +E* (T_cc1-T) ^F+D2* (T-T_cc3) ^F2

+ (A1 +B1 * T+C1 *T*Ln (T) +D1 *T²+E1 * (T_cc2-T) ^F1)

* (H1 * (1 -EXP ( - ( I1 *T) ^{Z *J1}) ) ) + (A2+B2* T+C2* T*Ln (T)

+D3 * T² +E2* (T_cc4-T) ^F3) * (H2* (1 -EXP ( - (I2*T) ²*^J2) ) )

The free energy near 0°C is small and positive so the enthalpy is dominated by the derivative of the free energy. Thus, since the free energy goes through a minimum the enthalpy must reverse its sign. The magnitude of the enthalpy actually is quite large at its extreme just below and above the free energy minimum. However, to estimate observable enthalpy one must use an equation that takes into account not only the molar enthalpy change of the protein in going from folded to unfolded, but also the proportion of protein molecules actually converting from one form to the other.

Because only a rather small proportion of the protein molecules are folding or unfolding anywhere in this temperature range, the observable enthalpy change is predicted to be small, less than +20 kJ/mole on heating between -15°C and 10°C. The positive enthalpy peak between -10°C and 0°C suggests that some stabilizing structural component is disassembling in that temperature range. Likewise, the negative enthalpy peak between 0°C and 10°C implies that refolding of the protein is accompanied by net bond formation.

The small enthalpy peak will occur at about 10°C upon heating because of the hysteresis shown in the raw data inset of this Figure. There is evidence in the literature (Tamura et al. Biochemistry 30, 11307, 1991; Antonino et al., PNAS 88, 7715, 1991; Griko et al FEBS Letters, 244, 2,276, 1989; Nakaya, M. et al. Biochemistry, 34, 3114, 1995) of several proteins showing a small positive enthalpy peak near 10°C.

The large transition at about 35°C is supported by our studies in various destabilizers, reported below.

All of these results lend support to a model in which the protein is systematically destabilized by exposure to temperatures approaching 0°C, but below 0°C the protein is restabilized.

EXAMPLE 2

Effects of Various Destabilizers

In the following presentation of the effects of various destabilizers and stabilizers, the raw ratio curves are presented with the baseline as an unprocessed representation of free energy. The fully stable and fully unfolded raw baselines are also shown. Positive deviations from the baseline in these solutions still indicate protein unfolding, but the signal is often composed of contributions from several different unfolded forms. Thus, the signal cannot be partitioned so as to represent the proportions of each of these moieties, and no mass action parameters derived. This means that the use of the ratio data from denaturants cannot properly be used to represent the free energy of unfolding of a single form of the protein by temperature alone. Figure 2a shows the fluorescence ratio signal as a function of the pH over the range from 8 to 13, with pH 2 as a highly stable reference. This protein is notable for its stability at low pH, but it becomes highly unstable in alkaline solutions. In alkaline solution, the ratio signal attains much larger values than the maximum seen at pH 2, which implies an increased extent of unfolding throughout the temperature range. In addition, the protein is unable to completely refold at intermediate temperatures where it is most stable at low pH.

At subambient temperatures and pH≥8 the protein is strongly destabilized. The unfolding is monotonically increasing as temperature is lowered and pH increases up to pH 12, although there is a significant reduction of signal above pH 12. In most cases, just as in the case of the partial unfolding at pH 2, there is a significant hysteresis (i.e., significant return to the initial state) upon warming. However, unlike the behavior in acidic conditions, the protein becomes relatively more unstable as it is warmed, showing no return to its original state over the time course of these experiments (5-6 hours). In these circumstances, the protein may unfold to a more open form at low temperature which may even be the more stable than the denatured form normally formed in never-cooled protein at intermediate temperatures where the protein is maximally stable. In general, the more open form appears not to be more stable at low pH in this protein since prolonged exposure of previously cooled protein to ambient conditions results in a return to the fluorescence ratio baseline over several days. The minima in these curves strongly support the existence of a stability maximum at about 35°C, and the slopes of the curves provide further support for the greater steepness of the decrease in stability in the direction of cooling, both of these observations as predicted by the theoretical curve shown in Figure 1b.

In the intermediate pH range 5.8 to 7.8 the protein manifests a complex unfolding behavior, as shown in Figure 2b. At temperatures below 20°C there is also a progressive destabilization with increasing pH and falling temperature. However, at low temperature there are clearly two separate unfolding processes, one the only process discernable at pH 2, and another, probably associated with a second domain, which is monotonically destabilized as pH increases and temperature decreases. It is remarkable that the unfolding-refolding dynamic associated seen at pH 2 appears to be very insensitive to pH over this range. Examination of the curves shows that both peak position and magnitude for this process remain virtually unchanged in both cooling and warming scans. Thus, whatever process is restabilizing the portion of the protein destabilized at 0°C in pH 2 solution, it is largely unaffected by these pH changes, as is the process causing the hysteresis. Another important observation is that the portion of the protein destabilized in high pH at low temperature is quite stable at temperatures as low as -20°C at pH 2. Thus it is not the effect of temperature alone which is destabilizing at temperatures well below 0°C.

EXAMPLE 3

Effects of Various Concentrations of Urea and Guanidine Hydrochloride on the β-lactoglobulin Cold and Heat Denaturation

In urea at concentrations above 2.0 M and GndHCl above 2.0 M, the protein is not able to fully refold at any temperature. As in the case of high pH, the nature of the curves at high concentrations of denaturant also supports the contention that there are two separate unfolding domains. This can be seen by examining the extensive refolding signal at intermediate temperatures for urea in the range 6 to 10 M and GndHCl in the range 3.5 to 7 M. In all of these cases, the signal level at maximal stability is higher than that of the fully heat denatured protein at pH 2, implying that all of the refolding occurs in another domain.

The low temperature data for urea at relatively low concentration, Figure 3a, are consistent with the model of water as a stabilizer as the temperature is lowered. It can be seen that at concentrations of urea as high as 1.0 M the ratio curves back towards the baseline, but as the concentration of urea rises the temperature of maximum denaturation is shifted to lower temperatures. This is despite the overall increase of denaturation efficiency as temperature falls due to more effective binding of the denaturant. This implies that falling temperature increases the magnitude of an unknown stabilization interaction, allowing the protein to refold despite increasing urea concentration, if the temperature is sufficiently low. Since the magnitude of maximal unfolding appears to increase monotonically with urea concentration, it can be inferred that the urea has at most a small effect on the stabilization interaction, instead the effects appear to be additive.

The effects of GndHCl at low temperature and relatively low denaturant concentration, Figure 3b, contrast sharply with the observations in urea, even though the data at high GndHCl concentrations asymptote to the same ratio value. Three concentrations of GndHCl below 0.5 M were investigated and the raw data are displayed in this figure. A 0.24 M GndHCl solution significantly stabilizes the protein during cooling by two criteria: lowering the maximum magnitude of the ratio, and shifting the maximum to a lower temperature. It was surprising to observe that raising the concentration of this powerful denaturant to 0.30 M further stabilized the protein during cooling, suppressing the size of the maximum ratio change by a factor of two. A further increase of GndHCl concentration to 0.42 M increases instability slightly because the maximum ratio excursion increases again, as well as the temperature of the peak maximum. However, the magnitude of the peak maximum is still smaller than in 0.24 M GndHCl. These results are in marked contrast to the effects of high pH (Figure 2b), which does not appear to effect either the magnitude of the maximum ratio excursion or the position of the maximum, and the influence of low concentrations of urea, which is generally destabilizing. This implies at least two independent mechanisms of interaction of GndHCl with the protein, perhaps the classic binding interaction (destabilizing) and an unknown stabilizing interaction. The magnitude and peak position of redenaturation and subsequent renaturation during warming, which is shown in Figure 3b for 0.30 and 0.42 M GndHCl is also noted. The magnitude of the ratio change is about equal for both concentrations and considerably larger than in 0.1 M NaCl buffer alone. Both GndHCl influenced transitions occur at lower temperatures than that of 0.1 M NaCl, with the 0.42 M being the lowest. The magnitude of the rewarming peak in both cases is much larger than that of the cooling peak, whereas in pH 7.5 the peaks are equal, and in 0.1 M NaCl the warming peak is noticeably smaller. The importance of these results for this invention is that they show that even a compound which appears to stabilize at 0°C is capable of increasing destabilization at higher temperature.

EXAMPLE 4

Effects of Slow Freezing and Thawing in Concentrated Urea Solutions

Figures 4a and 4b show the results of slow freezing and thawing respectively in concentrated urea solutions. The light scattering signal from the frozen samples precluded the use of the fluorescent ratio technique using a standard spectrofluorimeter, but semi- quantitative data can be obtained in these circumstances using the shift of the wavelength at the maximum (peak) intensity of fluorescence emission. For native β-lactoglobulin this is 335 nm, while the fully unfolded protein displays a peak at 350 nm. At the beginning of the cooling experiment, at 20°C, in 3 M urea the peak is not detectably red-shifted, that is destabilized, by one nm, the limit of resolution in these experiments. In 4 M it is red-shifted by one to two nm. The protein in 3 M solution shows no detectable change with respect to the control in 0.1 M NaCl until the temperature falls below 10°C, whereas in 4 M solution the peak position begins to detectably deviate from 336 nm below 18°C. The signal in 3 M is unable to attain the red shift of the 8 M solution even at -15°C, whereupon it froze. In contrast, the signal in 4 M solution overlaps that in 8 M solution at temperatures below -8°C. These results are remarkably consistent with actual ratio measurements as shown in Figure 3a, although examination of the ratio data shows that it is much more sensitive to small changes in protein stability ,e.g. the 3 M ratio data depart from baseline below 20°C. Immediately upon freezing, all signals blue-shifted precipitously, the 8 M to 339 nm, the 3 and 4 M to 336-337 nm. As startling as these results might seem, they can be explained by noting that the eutectic of urea is -17.62 ± 0.7°C at a composition of 8.2 M. Thus, the urea is first briefly concentrated to the eutectic concentration, then almost completely removed from the solution by precipitation. Simultaneously, the stabilizing salt, NaCl, is concentrated allowing the protein to largely refold despite the extreme viscosity. This is excellent evidence that the protein is thermodynamically, not just kinetically, stable at -35°C in concentrated NaCl. Data (not shown) from solutions of β-lactoglobulin in 2 M NaCl, 2 M urea supercooled to -28°C or 4 M NaCl supercooled to -35°C also displayed no detectable denaturation. Figure 4b shows the behavior of these samples during thawing. Unlike the case of freezing, all of the peak positions begin to red-shift at about-18°C, the eutectic point, although there is a peculiar lag in the shift in the 8 M solution. By -12°C the protein is once again denatured in the three solutions. This is not surprising since all of the ice melts by -12°C in the 8 M solution, and a large proportion of it melts in the 3 and

4 M solutions by -12°C. Thus, the NaCl is diluted to levels which cannot significantly compete with the urea and prevent denaturation. Nevertheless, the return to exactly the same red shift seen in this temperature range on cooling provides strong evidence that unfolding and refolding of this protein is independent of the presence of ice, contrary to many claims in the literature.

EXAMPLE 5

Effect of Recognized Stabilizers of

Fluorescence Ratio

Figures 5a and 5b show results of experiments analyzing the effect of recognized stabilizers on the fluorescence ratio of β-lactoglobulin at pH 2. Figure 5b shows the effect of various concentrations of glycerol on the denaturation of β-lactoglobulin. Figure 5a shows the effect of various levels of electrolyte.

The results (Figure 5b) at high temperature are remarkable in that there is no evidence for glycerol being a stabilizer. On the contrary, above 70°C there is a small gradual increase in the ratio with increasing glycerol concentration. Since the effect of glycerol itself on the ratio of free tryptophan is unknown it cannot be presumed that this small change represents a calculable change in the free energy, but the implication is that the T_den is almost unchanged and the rising slope of the ratio with increasing glycerol concentration would lead to a prediction of higher enthalpy. These predictions are borne out by DSC results presented below.

In contrast to the indifferent ability of glycerol to stabilize at high temperature, it displays effective stabilization at low temperature. At the relatively low concentration of 0.25 M a denaturation-renaturation peak is still seen, but its magnitude is attenuated and the T_max is about 5°C below that seen in 0.1 M NaCl alone. At 0.5 M. glycerol and above, the transition becomes undetectable. Extraordinarily, the ratio signal for all concentrations above 0.25 M are nearly overlapping each other, with only a small decrease in ratio implying additional stabilization from added glycerol. Even in 0.25 M, at the conclusion of the renaturation at -10°C, the signal returns to the level seen at higher concentrations. All of these levels are distinguishably below that of 0.1 M NaCl alone, showing that significant stabilization is achieved at relatively low glycerol levels.

Figure 5a displays fluorescent ratio curves in the presence of 0.1 M NaH₂PO₄, 0.05 M Na₂SO₄, or various concentrations of NaCl. Unlike glycerol, several of these solutions are strong stabilizers at high temperature, 0.05 M sulfate, in particular, appears to raise the midpoint of the transition more than 10°C above that in 0.1 M NaCl. These stabilizing effects can be verified with calorimetry. Published data for β-lactoglobulin in 0.1 M phosphate (Griko, Y. and Privalov, P. Biochemistry, 31,8810,1992), showing a T_m of 85°C, are in very close agreement with the phosphate curve on this graph. The NaCl solutions show a systematic stabilization with increasing salt concentration. The presumption of stabilization is based on the increase in T_m, although the reduction in ratio maxima is fully consistent with this picture.

At low temperatures the order of stabilization strength is essentially identical to that at high temperature except that the measure of stabilization is the extent of depression of ratio maxima, rather than a shift in T_m. The peak positions stay constant despite suppression of peak magnitudes. Inasmuch as the raw ratio curves accurately reflect the free energy of unfolding in these circumstances, this is contrary to the results predicted by the classical theory. In the classical view, an increase in the upper T_m is linked to a concurrent decrease in the temperature of unfolding at low temperature. These results show that the ordinary stabilizers used in industry and medicine to store proteins safely, such as ammonium sulfate, are in fact measurably effective as stabilizers when measured by fluorescence ratio. However, it is also important to note that there is still a detectable loss of stability in all of these solutions, which means that the extent of stabilization is very small, usually 5 to 10 kJ/mole. This emphasizes the importance of the present method since it is equivalent to saying that a significant level of destabilization is always seen in the β-lactoglobulin.

EXAMPLE 6

Extrinsic Fluorescence Ratio of

β-Lactoglobulin in ANS

In order to further confirm the veracity of the intrinsic fluorescence ratio measurements as an accurate representation of the free energy, the extrinsic fluorescence ratio signal from β-lactoglobulin in the presence of ANS was examined. This fluorophore binds most efficiently to hydrophobic sites that are normally relatively immobile in the native protein. When bound to such sites the fluorescence emission maximum of the ANS-protein complex is at 480 nm, whereas when free in solution it is at 510 nm. When the protein is denatured no red-shift in the peak maximum is observed, but a sharp reduction in intensity is seen. At 440 nm a relatively small change in intensity occurs as a result of denaturation, thus the ratio of intensities 480/440 is a good normalized estimate of unfolding. The result on supercooling to -12°C from 20°C, then rewarming to 95°C is shown in Figure 6a. The observed pattern is virtually identical to that of the intrinsic fluorescence ratio, including the hysteresis of the denaturation-renaturation process subsequent to cooling. Since the fluorophore is extrinsic, and global in its binding, the virtual identity of this curve with the intrinsic fluorescence curve strongly supports the view that the event at 0°C is destabilized protein, i.e., an increase in the proportion of denatured protein, not simply a change in the structure of the native state. Further support for the intrinsic fluorescence measurements was provided by analyzing the fluorescence emission of retinol, the putative natural substrate for this protein (Dufour et al, Bipolymers 33,589-598, 1993), at 480 nm as shown in Figure 6b. An increase in fluorescence intensity indicates increased binding. In this case the increased binding displays a strong temperature dependence. Thus, the significant fall in fluorescence signal over the wide temperature range between 0°C and 100°C is not indicative of a proportional increase in the fraction of unfolded protein, but instead represents a rapid increase in the dissociation constant with increasing temperature. Nevertheless, significant changes in binding affinity over the range of a few degrees, consistent with data from other fluorophores, can be taken as additional proof of a denaturation- renaturation process, which is what is seen in Figure 6b. Between about 10°C and 0°C during cooling the binding affinity drops sharply, then, just below 0°C it rises even more steeply to about -8°C, whereupon it increases much more gradually to -18°C. The warming record begins at 2°C, and the affinity drops at a constant rate until 5°C, whereupon it begins to decrease faster, implying increased unfolding of the protein. At 9°C the affinity suddenly begins a rapid increase until 13°C, consistent with refolding, then very slowly increases until 18°C, whence it begins its normal monotonic decrease with increasing temperature. The entire cooling and rewarming pattern below 20°C is in close agreement with the tryptophan and ANS data.

EXAMPLE 7

Calorimetric Studies

A typical β-lactoglobulin denaturation experiment in 0.1 M NaCl in the DASM calorimeter is shown in Figure 7a. The integrated enthalpy of the transition, and the position of the peak are consistent with literature values in other buffers. The average results from 29 experiments on DASM 1, DASM 4, and DASM 4 M calorimeters are <ΔH>=290.46±17.285 kJ/mole, <ΔT_m>=80.97± .607°C, <ΔH^cal/ΔH^vH>=1.053+.068. Thus, the transition appears to be two state by the traditional criteria, and the observed enthalpy is slightly higher than that calculated from the fluorimetric data (256 kJ/mole), which is nevertheless within two standard deviations of the calorimetric mean. It should be noted that the strongly decreasing signal at temperatures above 95°C is a general result in these experiments. The simplest explanation for this slope is aggregation of the denatured molecules. Inasmuch as aggregation takes place in the temperature range of the transition, it has the effect of reducing the magnitude of both ΔC_p and the excess ΔC_p. However, it is reasonable to presume that the aggregation occurs predominantly at temperatures above the peak of excess heat capacity where the majority of the molecules are unfolded. This leads to the conclusion that the dominant effect is reduction of the apparent heat capacity of the denatured state and, in turn, would skew the measured enthalpy to values greater than the actual value, especially under conditions where aggregation is favored such as high protein concentration. The mean enthalpy calculated from experiments in which the concentration was less than 0.80 mg/ml (11 out of 29) yielded 285 kJ/mole; from the balance of the experiments the mean enthalpy was 294 kJ/mole. The small peaks at

110°C and 125°C are consistent with the unfolding of a second, more stable, domain, being suppressed by the precipitation. The peak at 125°C has been shown by others

(Suttiprasit). The importance of these measurements is that they demonstrate that, in a situation in which fluorimetric data can be used to directly predict the thermodynamic quantities derived from calorimetric measurements, the results are very close.

In Figure 7b the results from a typical denaturation experiment in 0.1 M glycine buffer, pH 2, are presented (curves 1 to 1b), and compared to a denaturation experiment in which 2.0 M GnHCl has been added to the glycine (curve 2). On the basis of a nearly constant slope at temperatures above the end of the first denaturation peak, it appears that aggregation in glycine buffer is significantly inhibited in solutions containing less than 0.5 mg/ml β-lactoglobulin. The general picture is similar to 0.1 M NaCl at temperatures below 95°C. For 14 experiments <ΔH>=293±15.5 kJ/mole, <ΔT_m>=79.75±0.35°C, and <ΔH^cal/ΔH^vH>=1.017±0.03. Thus the transition again appears to be two state, the enthalpy is indistinguishable from that in 0.1 M NaCl, and the protein appears to melt about 1°C lower. At temperatures above 95°C, however, several unique characteristics can be seen. The apparent ΔC_p is about 9-10 kJ/mole, larger than it is normally thought to be (Griko, Y. and Privalov, P. Biochemistry 31, 8810, 1992; Griko et al, Biophysical J. 1994; and with the theory embodied here). Above 118°C a significant endotherm is apparent (Figure 7B, left panel, curves 1-16), which is incomplete by the measurement limit at 130°C, but which is consistent with the unfolding of a second domain observed by others (Sukttiprasit) and indicated by the fluorescence results described herein. The second and third heating scans are shown here as well and they indicate that whatever damage the protein incurs in the domain which initially unfolds at 80°C the heat capacity remains nearly the same as immediately after the initial denaturation. Furthermore, upon reheating up to two times from low temperature it can be seen that the endotherm about 115°C remains intact, indicating that the second domain is highly heat stable.

The addition of 2.0 M GndHCl to the glycine buffer

(curve 2) is instructive because it destabilizes both domains without eliminating the separation of unfolding events as a function of temperature. The deconvolution of the excess ΔC_p with and without GndHCl is shown in the right panel of Figure 7b. Given that the deconvoluted first domain peak in GndHCl is shifted down 13°C, it is remarkable that the upper peak is shifted from some temperature above 130°C to 95°C. These results support the existence of two separate cooperatively unfolding domains. This is what is implied by the fluorescence destabilization studies, (Figures 2 and 3). Thus the fluorescence ratio measurements are sensitive to this level of complexity.

Figure 7c shows the effect of increasing glycerol concentrations in 0.1 M NaCl on the excess ΔC_p of high temperature denaturation. There is no detectable shift in <ΔT_m>, which is 81.37±0.25°C compared to the <ΔT_m> in 0.1 M NaCl alone, 80.97±.607°C. In contrast, the enthalpy increases monotonically with the glycerol concentration. At 5.0 M glycerol it reaches 355.6 kJ/mole. Thus, these results are in direct contradiction to the Timasheff size- exclusion hypothesis. Instead, this means that the net entropy change during unfolding is positive i.e. breaking solution structure. This strongly implies that the protein stabilization at low temperature is due to the large enthalpy debt incurred in breaking solution structure. In turn, this implies that the degree of stabilization for most globular proteins which do not directly bind the stabilizer will be about the same.

Figure 8 shows the low temperature behavior of concentrated β-lactoglobulin solution as measured in a Perkin-Elmer DSC 4. Even though the concentration is 100x that in the DASM experiments, the high temperature behavior (not shown) is virtually identical to, and completely consistent with data published by others (Relkin and Launay, 1990, Suttiprassit, 1992).

In the initial phase of the experiment the sample was supercooled to -14°C from +40°C and showed no evidence of exothermic behavior in the temperature range in which fluorimetry confirms that a moderate level of destabilization occurred. Subsequent to freezing, cooling continued to -160°C. No significant events were observed. These results are poorly correlated with the predictions of enthalpy and T_m given by classical theory, -243 kJ/mole and about -23°C as calculated using the published ΔH, T_m, and ΔC_p for high temperature denaturation, 312.2 kJ mole^-1, 78°C, and 5.58 kJ mole^-1 deg^-1 respectively (Griko and Kutyshenko, 1994, Biophysical J). From -160°C the sample was rapidly rewarmed to -2°C, then recooled again to -40°C. The purpose of this second cooling trace was to insure that the significant denaturation signal was not accidentally hidden by the sudden freezing of the supercooled water. No detectable transition was found.

EXAMPLE 8

Circular Dichroism

Figure 9 shows the results of a far UV circular dichroism study of the denaturation of β-lactoglobulin by extreme temperatures and high concentrations of denaturants. Also included are curves from the literature which are thought to represent highly disordered structure. The addition of either urea or GndHCl in high concentration to the β-lactoglobulin at subzero temperatures produces a significant positive shift in the ellipticity, measured at wavelengths longer than 210 nm, such that the signal displayed closely approximates that recorded at 10°C from the completely random coil synthetic polypeptide Glu-Lys-Lys-Leu-Glu-Gln-Ala (SEQ ID NO:1). As shown in Figures 3a and 3b the fluorescent signal in high denaturant at temperatures below -5°C implies almost complete unfolding consistent with the CD. However, the synthetic peptide displays a large change in CD pattern in going from 10°C to 80°C. This implies that the random coil does order itself in some way at low temperatures. At high temperatures and high concentrations of denaturant the β- lactoglobulin CD signal is close to, but remains somewhat more negative than that of the artificial polypeptide at high temperature (Privalov et al, J. Mol. Biol. 1989). This indicates that there is considerably more residual structure left in the protein, in the absence of denaturants, at high temperature than in the synthetic polypeptide. The fluorescent ratio signal under these conditions (Figures 3a and 3b) is consistent with the view that considerable native state structure is retained at high temperature, because, as pointed out previously, at high temperatures the difference between the signal from free tryptophan plus denaturant and protein plus denaturant is larger than that at subzero temperatures. All of these results imply that unfolding of the protein at low temperature is more complete, but also that more local ordering takes place. This increase in order is a likely source of the renaturation hysteresis seen after reheating from a temperature ≥ 0°C. to ambient or above.

Figure 10a shows the temperature dependence of β-lactoglobulin CD spectral data in 0.1 M NaCl in the far UV region. All spectra except the initial 25°C spectrum are subsequent to cooling from 25°C to -0.5°C. It is clear that at 65°C the spectrum is shifted towards the 95°C spectrum in what appears to be a consistent proportion throughout, and that the 40°C and 25°C spectra on rewarming are very close to the initial 25°C run. In contrast, it can be seen that the spectrum taken at -0.5°C displays a positive ellipticity relative to the initial spectrum at wavelengths longer than 237 nm while at shorter wavelengths the ellipticity is negative. In the standard approach these spectra would be deconvoluted using a basis set such as that used by the Lincomb program

(Perczel, Park, and Fasman, Anal Biochem, 1992). For both the initial 25°C record and for the 95°C record this is a reasonable calculation because it can be assumed that in these cases virtually all of the molecules are in the native or denatured states, respectively, whereas at the other temperatures there is a mixture of both states, making ordinary deconvolution highly inaccurate. For the native state prior to cooling, the deconvolution yields 22% random coil, 26% β sheet, 13% α helix, 10% β turn, and 29% aromatic, with an r² of 0.988 (Papas et al, Nature, 1986). The aromatic signal can reasonably be partitioned into (1) the sum of the aromatic residues plus the disulfide bonds; and (2) the β barrel structure known to be present in this protein. If that part of the aromatic signal in excess of the sum of disulfide bridges and aromatic residues is then combined with the β sheet estimate to give an effective estimate of total β sheet structure this yields a modified deconvolution very close to the X-ray data of 51.2% β sheet (modified CD estimate = 47.6%), 10.5% β turn, 6.8% α helix, 7.4% aromatics, and 24.1% random coil (Papas et al, Nature, 1986, Monaco et al, JMB 1987). The deconvolution of the 95°C signal cannot, of course, be compared to X-ray data, but the high r² of 0.987 leads one to believe it is a reasonably good measure of structure. The fit yields 16% β sheet, 7% β turn, 9% α helix, 15% aromatics, and 53% random coil. By the same reasoning as above, this would lead to real estimate of β sheet of 24%.

To circumvent the problem of analyzing spectra in which both native and denatured states make significant contributions, the 25°C spectrum was used first, prior to cooling, and the 95°C spectrum as an intrinsic basis set (IBS). Optimizing a linear combinations of these vectors one can fit the observed spectra far better than using artificial basis state vectors. Nevertheless, the DS theory predicts (see Figure 1) that ΔG_den is about 8.2 kJ/mole at 25°C on cooling. This means that a small but significant proportion of molecules is denatured under these circumstances (about 3.5%). To correct for this applicants subtracted 0.035 times the 95°C spectrum at each point, then renormalized by dividing by 0.965 to obtain an estimated pure native state signal at 25°C. This was also done at 95°C. where the DS curve predicts a free energy of -12.3 kJ/mole which translates into 1.7% native state. By exhaustively searching the space of the linear combinations of these vectors, the percentage of native state and denatured signal extracted from a minimum least square fit to spectra taken at other temperatures yields a direct estimate of the ΔG_den as in the case of fluorescence ratio measurements. The IBS deconvolution of the 40°C data, as shown in Figure 10c, gives a nearly perfect fit (r²=0.9972), 5.0% denatured sate, 95.0% native state indicating a nearly complete return to the initial state at 25°C (Figure 10a). This proportion of denatured molecules translates into a free energy of unfolding of 7.3 kJ/mole, which is extremely close to the fluorimetric estimate of 7.04 kJ/mole upon rewarming (see Figure 2).

A significant further improvement in fit can be achieved in all of the cases considered here when a variable fraction of each of a set of expanded and modified Fasman basis state vectors is independently subtracted or added to each the denatured and native experimental basis vectors (IBS). For each such change an optimal linear combination of the modified basis vectors

(MIBS) is calculated. Thus, the global best fit is the linear combination having the smallest sum of squares after an exhaustive search of the space of all such modifications of the denatured state vector. In addition to providing a more likely estimate of the free energy, this method estimates the proportions of the secondary structural changes distinguishing the test states from the reference states.

Starting with the spectrum at -0.5°C, the difference between MIBS and IBS is illustrated by the respective r² values: IBS=0.98978, MIBS=0.99987 (Fig.10b curves 1 and 3). At 40°C r² (IBS) =0.99764 (Fig. 10c curves 1 and 2) whereas r² (MIBS) =0.999972 (Fig. 10c curves 1 and 3). At 65°C r² (IBS) =0.99867) (Fig. 10d curves 1 and 2) whereas r² (MIBS) =0.999972 (Fig. 10d curves 1 and 3). Taking the MIBS deconvolution as the most accurate representation of the physical changes in the protein allows further comparison of the estimated free energy of unfolding with fluorescence ratio data. At -0.5°C MIBS gives 1.5 kJ/mole, fluorimetry 2.93 kJ/mole, at 40°C, 10.6 kJ/mole versus 6.89 kJ/mole; and at 65°C, 4 kJ/mole versus 7.58 kJ/mole. These values are in reasonable agreement as can be seen by comparing the percentage of denatured and native protein predicted by the two methods at each temperature. At -0.5°C MIBS predicts that the protein is about 34% unfolded. This compares very favorably to the value from the DS theory of 27.9% fraction unfolded. At 40°C MIBS predicts 1.7% fraction unfolded whereas DS theory predicts 7% fraction unfolded. At 65°C MIBS predicts 19.5% fraction unfolded whereas DS theory predicts 6.7% fraction unfolded. These differences in the estimates of the free energy between the two methods may be accounted for by assuming that the more global measure of the circular dichroism technique allows more accurate recognition of global structural changes in both native and denatured protein states. In this hypothesis what is implicitly assumed is that the intrinsic of the tryptophan in each form is dependant on the global structure of the form. Thus even though the percentage of the form might no vary the fluorescence signal could vary giving the appearance of the variation of the proportion of each form. On the other hand it is reasonable to assume that in any given case the fluorescence is the more accurate measurement because the proturbation vectors that are used to modified the shape of the intrinsing basis vectors are of unknown accuracy in representing the actual structural forms in the native and denatured state at each temperature. Thus a more accurate set of perturbation vectors (which are not currently available) by modifying each of the intrinsic vectors in a different way might lead to a proportion of modified native and denatured states much closer to the estimate of the fluorescence measurement. Nevertheless in the crucial temperature range from 0°C to physiological temperatures the predictions by both methods are in good agreement and mutually supportive.

EXAMPLE 9

Fluorescence Ratio Data

from Other Proteins

The β-lactoglobulin results are compelling in a general sense only if they represent nearly universal behavior in globular proteins. Figures 11-13 are proof of the ubiquity of the destabilization-restabilization dynamic seen in the β-lactoglobulin. Figure 11 displays a comparison of β-lactoglobulin (panel 2) with horse skeletal muscle myoglobin. In panel 1 the ratio signal of the myoglobin at ph 4.8 is shown. It appears that by -10°C about 50% of the protein has unfolded. This is remarkable because published calorimetric data on this protein under these conditions indicates no detectable signal (Privalov, Griko, Venyaminov, and Kutyshenko, J Mol. Biol., 1986). This emphasizes the fact that if the enthalpy of the transition is rather small even a substantial conversion of native to denatured form will yield a weak calorimetric signal. It should also be noted that there are apparently at least two denaturation processes illustrated in panel 1. The widespread denaturation- renaturation process between 10°C and -10°C during cooling which has been exhaustively characterized herein by β- lactoglobulin data (panel 2 and Figures 1 -10) appears to be superimposed on a much more massive denaturation similar to the β-lactoglobulin data at pH 7-9 (Figures 2a,b). It is likewise important to note that there is a significant hysteresis during rewarming, which persists through the high temperature denaturation.

Panel 2 represent ratio curves for β-lactoglobulin which has been exhaustively examined in figures 1 to 10. However, in this figure additional data are included in the inset showing significant destabilization of the protein after prolonged storage at 0°C. It can be seen that throughout the temperature range of the analysis the annealed protein is significantly destabilized compared to the non annealed protein as shown in the main body of the panel. In addition prolonged storage at 0°C has introduced at least two additional denaturation-renaturation events between 10 and -10°C. This is especially important to protein formulations with complex freeze-drying or low temperature histories. In these cases these results make clear that temperature changes of less than 5°C can make a large difference in protein stability during sample preparation or storage. This can be understood more clearly by referring to figure 1 the DS theory free energy curve for unannealed β-lactoglobulin. At the temperature of maximum instability near 0°C the ΔG_den is about +3 kJ/mol which corresponds to about 28% of the protein being denatured. Referring back to the insert in Fig. 11 panel 2, below -11°C the proportion of unfolded molecules approaches 40%, but at -6°C it is about 20%. The key concept in these cases is that in situation where the ΔG of unfolding is near zero, very modest changes in free energy reflect large changes in the proportion of molecules unfolded. It is the fraction of protein unfolded which determines the ability of cells to recover from cold shock injury or the acceptability of pharmaceutical preparations.

In panel 3 the myoglobin data at pH 7 are shown. This is a pH range wherein the protein is stable. The fluorescent ratio data are consistent with this view, but significant denaturation is still detectable below 10°C. In addition, the right side of panel 3 shows that even at pH 7 there is a significant increase in denatured protein in the range 60 to 70°C subsequent to cooling as compared to protein heated from room temperature. Thus, there is good evidence for persistent destabilization subsequent to cooling even at pH 7.

In Figure 12 data for four more representative proteins is shown.

Panel 1 shows the fluorescence ratio scan of actin, with the inset showing the integrated fluorescence intensity from 320 nm to 350 nm in 10 nm steps. In this case the fluorescence ratio is unusually insensitive to the denaturation-renaturation event near 0°C. Examination of the full spectra (not shown) make it clear that this is the result of a very unusual situation in which there is virtually no change in the spectral symmetry upon unfolding over a wide range of wavelengths. In this case use of a very low wavelength, near 305 nm for a divisor would produce a ratio curve similar in structure to that of β-lactoglobulin. Nevertheless, the integrated intensity clearly shows the usual low temperature behavior. Both the integrated intensity and ratio are consistent in showing that cooled protein remains significantly destabilized at high temperature.

Panel 2 shows the fluorescence ratio data for human plasma fibronectin, taken at pH 7 where it is known to be very stable. The inset emphasizes the details of low temperature cooling and rewarming behavior. The pattern throughout is remarkably similar to β-lactoglobulin, the only notable difference being that at -10°C the fibronectin appears to reach maximum stability. It is thus noteworthy that, despite the apparent subzero stabilization, destabilization is apparent from +5°C to the threshold of the heat denaturation.

Panel 3 shows the fluorescence ratio data for trypsin at pH 4.2, near its maximum stability point. The behavior is remarkably similar to that of myoglobin at pH 7, and like all the other proteins shown here destabilization subsequent to exposure to cold persists up to the heat denaturation temperature. As in the case of myoglobin, the denaturation-renaturation events near 0°C seem superimposed on a larger, more general cold destabilization which is monotonically increasing with decreasing temperature below ambient. Comparison of this behavior with the low pH myoglobin behavior (Figure 11, panel 1) and the high pH β-lactoglobulin behavior (Figures 2a,b) implies that this is the result of unshielded electrostatic forces increasing as the temperature falls. This destabilization process is not confined to the temperature range in, the immediate vicinity of 0°C, but under destabilizing conditions it would appear to be sharply potentiated by temperatures at or below 0°C. This implies that the process which leads to the denaturation- renaturation near 0°C occurs in both hydrophobic regions and regions having a significant admixture of charged and hydrophobic residues. In the latter case the transition may frequently lead to a sharp decrease in charge separation and a consequent loss of stability (cold denaturation). Panel 4 shows the fluorescence ratio analysis of α chymotrypsin at pH 4.2 at which pH the protein is known to be very stable. The behavior of the protein is virtually identical to that of β-lactoglobulin and fibronectin throughout the temperature range. An additional curve is included, labeled 'post-freeze reheating'. This shows clearly that freezing greatly exacerbates the persistent instability during rewarming from 5°C up to and including the heat denaturation.

Figure 13 panels 1 to 4 show ratio data for two additional model proteins.

Panel 1 shows the unusual fluorescence ratio trace from cooled bovine serum albumin (BSA). The mammalian serum albumins are known to have tryptophans in the hydrophobic patches on the surface of the protein

(Permyakov, E. 1993 Luminescent spectroscopy of proteins) and during unfolding these tryptophans are moved in more hydrophobic environment. Thus, the ratio signal for denaturation is negative in these proteins. This provides an important test of the representations of the low temperature ratio signal in the present embodiments. As this panel shows all of the low temperature signal are of the opposite sign to those displayed by the other proteins in the previous figures. In contrast the structure of the changes are virtually identical. This provides strong evidence that the low temperature signal truly represents denaturation-renaturation events. Furthermore, this shows even in the case of very large multi-domain polypeptides (MW 65000) the transitions between 10 and -10°C have a simple and consistent interpretation. This provides further support to the idea that the physics determining this low temperature transition as a consequence of the general chemistry of the local secondary structure of these proteins, probably of that of hydrophobic regions.

Another important implication of panel 1 is that the destabilization at 0°C is independent in size and complexity and can be monitored in protein where the tryptophans are moved from the interior of the protein to the aqueous environment or vice versa.

Even though the inverse albumin signals might imply that cellular signal will not be clearly defined the predominance of the positive going denaturation signal will be dominant as illustrated be other proteins shown here. In contrast the additional evidence that panel 1 presents for the ubiquity of the denaturation at 0°C is further support for the clinical applicability of hypothermia for treatment of malignancies.

It is also important to note that below -5°C the BSA appears to enter into a second denaturation. As in the case of annealed β-lactoglobulin (Fig.11 panel 2) this is another example of the existence of multiple denaturation- renaturation events below 0°C.

Panels 2 to 4 show the fluorescence signal ratio for lysozyme over a range of pH and with and without the non hydrolyzable substrate chitotriose.

Panel 2 shows lysozyme in acetate buffer pH 4.8 in the absence of substrate. At this pH it is known that two of the three tryptophans close to the active binding side of the enzyme are unquenched. The general pattern of the ratio curves is similar to β-lactoglobulin. The apparent level of cold induced instability during cooling and subsequent reheating appears to be grater than that of β- lactoglobulin. The curves were generated using nearly the same wavelengths as those used to generate the ratio curves for enzyme-chitotriose complex (panels 3 and 4). This allows the direct quantitative comparison of the transitions between the bound and unbound enzyme. The insert shows the ratio for unbound enzyme for optimal set of wavelengths.

Panel 3 shows lysozyme in excess of chitotriose at pH of maximum enzyme activity (5.5). As in the case of the uncomplexed enzyme pH 4.8 one of the three tryptophans near the active side is unquenched. Panel 4 shows the same enzyme complexed with the same substrate at pH 7.5 where all of the tryptophans near the active side are unquenched.

The effect on the chitotriose on the ratio signal is large and fully supportive of the interpretation of the low temperature events as denaturation-renaturation. It should be noted especially that native lysozyme complexed with the substrate displays a much lower ratio than uncomplexed native lysozyme whereas the denatured protein shows precisely the same ratio independently of the presence of the substrate or pH. This makes the negative going ratio signal below 0°C in the presence of chitotriose unambiguously that of denatured protein renaturing. To see this it is instructive to postulate that the signal represents a local structure of the denatured molecules which removes the tryptophans from the aqueous solution as it is supercooled. The decreasing signal would then be a consequence of the decreasing signal of the unfolded fraction of the protein. However, the denatured molecules are insensitive to the presence of chitoteriose thus this putative change in the denaturation ratio will be operand in the solution of lysozyme at pH 4.8 without substrate panel 2. From panel 2 it can be approximately calculated that the change of the ratio of the denatured state will be of the magnitude of 0.06. This in turn would produce a negative shift in ratio of about 0.01 on panel 3. The observed negative shift is in order of magnitude larger. Thus the argument that the negative going signal can be explained largely by restructuring of the denatured state is untenable.

The lysozyme chitotriose study provides further evidence of the veracity of the fluorescence ratio method of determining protein stability by two important criteria. At temperatures below 35°C in the absence of substrate at pH 4.8 the enzyme becomes monotonically destabilized with decreasing the temperature to 0°C. In the presence of chitotriose the destabilization commences only below 20°C. Thus at low temperatures the binding of the substrate stabilizes the enzyme as would be expected. Even though high temperature would be expected to inhibit the binding of substrate, to the extend that binding is still significant at the denaturation temperature, chitotriose would be expected to stabilize the protein against the denaturation as well. As can be seen from a comparison of panel 3 and 4 with panel 2 there is a shift of +5°C in temperatures of both the predenaturation event and the heat denaturation in the presence of substrate.

In panel 4 it should be noted that chitotriose- lysozyme complex at pH 7 provides further evidence for multiple denaturation-renaturation events at subzero temperatures in aqueous protein solutions.

A significant result of fluorescence ratio analysis of lysozyme chitotriose complex at pH 5.5 and pH 7.5 is the indication that the degree of maximum denaturation near 0°C and the extent of denaturation hysteresis during reheating are not necessary positively correlated. Examination of the ratio curve at pH 5.5 indicates that the maximal instability at 0°C is greater than the maximal instability at 0°C at pH 7.5. In contrast comparing the differences between the heating and reheating curves at both pH values shows that throughout the range from 20 to about 55°C the degree of protein destabilization is greater at pH 7.5.

All publications mentioned or cited above are hereby incorporated by reference. The scope of the presently disclosed invention is not to be limited by the above detailed description of certain preferred embodiments. It will be apparent to those skilled in the art that various modifications and equivalents of the disclosed invention can be made without departing from the spirit or scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of evaluating agents capable of enhancing cellular instability comprising :

(a) providing a first sample of cells of a single cell type;

(b) determining a range of stress over which said cells are viable;

(c) measuring within said range of viability the fluorescence emission spectra of said cells in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a first emission spectrum;

(d) providing a second sample of said cells of said single cell type wherein the proteins of said cells are substantially denatured;

(e) measuring the fluorescence emission spectra of said cells of said second sample in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a second emission spectrum;

(f) comparing said first and second spectra to determine lambda (1) and lambda (2), such that lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between said first and second spectra;

(g) providing a third sample of said cells of said single cell type;

(h) determining a control plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said third sample over a range of increasing stress followed by a range of decreasing stress, wherein said increasing stress produces protein instability; (i) providing a fourth sample of said cells of a single cell type;

(j) adding said agent to said fourth sample to produce a test sample; (k) determining a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said test sample over a range of increasing stress followed by a range of decreasing stress, as in step (h); and

(l) evaluating the ability of said agent to enhance cellular specific instability by comparing said control and said test plurality of ratios, such that instability will be increased where said control plurality of ratios is greater than said test plurality of ratios for any given amount of stress in at least one of the increasing or decreasing directions.

2. A method of evaluating an agent for enhancing cellular instability comprising

(a) providing a sample of cells of a single cell type;

(b) adding said agent to said sample to produce a test sample;

(c) determining a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said test sample over a range of increasing stress followed by a range of decreasing stress, wherein said increasing stress produces protein instability, and lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between a first spectra and a second spectra, said first spectra is a fluorescence emission spectra of a first sample of said cells in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm within a range of applied stress where said cells are viable and said second spectra is a fluorescence emission spectra of said cells in the range of 300-500 nm when excited by a light beam in the range of 270- 700 nm within a range of applied stress wherein the proteins of said cells are substantially denatured;

(d) evaluating the ability of said agent to enhance cellular specific instability by comparing said test plurality of ratios with a control plurality of ratios developed for cell sample without any agent, such that instability will be increased where said control plurality of ratios is greater than said test plurality of ratios for any given amount of stress in at least one of the increasing or decreasing directions.

3. A method of evaluating agents for enhancing cellular stability comprising:

(a) providing a first sample of cells of a single cell type;

(b) determining a range of stress over which said cells are viable;

(c) measuring within said range of viability the fluorescence emission spectra of said cells in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a first spectrum;

(d) providing a second sample of said cells of said single cell type wherein the proteins of said cells are substantially denatured;

(e) measuring the fluorescence emission spectra of said cells of said second sample in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm to provide a second spectrum; (f) comparing said first and second spectra to determine lambda (1) and lambda (2), such that lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between said first and second spectra;

(g) providing a third sample of said cells of said single cell type; (h) determining a control plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said third sample over a range of increasing stress followed by a range of decreasing stress, wherein said increasing stress produces protein instability;

(i) providing a fourth sample of said cells of a single cell type;

(j) adding said agent to said fourth sample to produce a test sample;

(k) determining a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said test sample over a range of increasing stress followed by a range of decreasing stress, as in step (h); and

(l) evaluating the ability of said agent to enhance cellular specific stability by comparing said control and said test plurality of ratios, such that stability will be increased where said control plurality of ratios is less than said test plurality of ratios for any given amount of stress in at least one of the increasing or decreasing directions.

4. A method of evaluating an agent for enhancing cellular stability comprising:

(a) providing a sample of cells of a single cell type;

(b) adding said agent to said sample to produce a test sample;

(c) determining a test plurality of ratios of fluorescence intensity at lambda (1) to fluorescence intensity at lambda (2) in said test sample over a range of increasing stress followed by a range of decreasing stress, wherein said increasing stress produces protein instability, and lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between a first spectra and a second spectra, said first spectra is a fluorescence emission spectra of a first sample of said cells in the range of 300-500 nm when excited by a light beam in the range of 270-700 nm within a range of applied stress where said cells are viable and said second spectra is a fluorescence emission spectra of said cells in the range of 300-500 nm when excited by a light beam in the range of 270- 700 nm within a range of applied stress wherein the proteins of said cells are substantially denatured;

(d) evaluating the ability of said agent to enhance cellular specific stability by comparing said test plurality of ratios with a control plurality of ratios developed for cell sample without any agent, such that stability will be increased where said control plurality of ratios is greater than said test plurality of ratios for any given amount of stress in at least one of the increasing or decreasing directions.

5. The method of any one of claims 1-4 wherein said excitation light beam has a wavelength of 280 nm.

6. The method of any one of claims 1-4 wherein said stress is at least one stress selected from the group consisting of decreasing temperature, increasing temperature, decreasing pH, increasing pH, increasing level of ionizing radiation and chemotherapeutic agent; and said single cell type is selected from the group consisting of malignant cells, cells derived from human tumor, cells derived from plant seeds, cells derived from germinating plant seeds, and cells derived from cryopreserved vertebrate tissue.

7. A method of evaluating agents for enhancing the stability of protein in a protein formulation comprising:

(a) providing a first sample of said protein formulation; (b) determining a range of temperature, at a constant stress, over which said protein remains stable;

(c) measuring the fluorescence emission spectrum in a range of 300 - 500 nm of said protein formulation when excited by a light beam in a range of 270-700 nm to provide a first spectrum, said measuring being performed at a temperature within the range determined in step (b);

(d) providing a second sample of said protein formulation wherein said protein is heat denatured at said constant stress;

(e) measuring the fluorescence emission spectrum in a range of 300-500 nm of said second sample when excited by a light beam in a range of 270-700 nm to provide a second spectrum, said measuring being performed at said constant stress;

(f) comparing said first and second spectra to determine lambda (1) and lambda (2), such that lambda (1) is the wavelength of maximum emission intensity difference and lambda (2) is the wavelength of minimum emission intensity difference between said first and second spectra;

(g) providing a third sample of said protein formulation such that said protein is maximally stabilized at said constant stress;

(h) repeatedly recording a fluorescence intensity at lambda (1) and a fluorescence intensity at lambda (2) and forming a ratio of fluorescence intensity at lambda (1) divided by a fluorescence intensity at lambda (2) of said third sample excited by a light beam in the range of 270-700 nm, while simultaneously sampling the temperature over said range of temperature as determined in step (b) providing a stable base line;

(i) providing a fourth sample of said protein formulation wherein said protein has irreversibly lost its activity at a temperature T, at said constant stress, such that said protein is not necessarily fully denatured over a temperature range of interest;

(j) repeatedly recording a fluorescence intensity at lambda (1) and a fluorescence intensity at lambda (2) and forming the ratio of fluorescence intensity at lambda (1) divided by the fluorescence intensity at lambda (2) of said fourth sample excited by a light beam in the range of 270-700 nm, while simultaneously cooling from a temperature T, to a lower temperature of interest, wherein said protein is maximally destabilized, providing an unstable base line;

(k) providing a fifth sample of said protein formulation as a control sample;

(l) repeatedly recording the fluorescence intensity at lambda (1) and the fluorescence intensity at lambda (2) and forming the ratio of the fluorescence intensity at lambda (1) divided by the fluorescence intensity at lambda (2) of the said fifth sample of the protein formulation excited by a light beam in the range of 270-700 nm at said constant stress, while scanning temperature over a temperature range of interest providing a control experimental ratio curve;

(m) providing a sixth sample comprising said protein formulation and an agent to be tested as a stabilizer;

(n) determining a test plurality of ratios of fluorescence intensity at lambda (2) to fluorescence intensity at lambda (1) in said sixth sample while the temperature is simultaneously scanned over said temperature range of interest, providing a test ratio curve;

(o) evaluating the ability of said agent to enhance stability of said protein formulation by comparing said recorded ratios of said fifth sample (control) and said test plurality of ratios of said sixth sample (test), such that for any given temperature, stability will be increased whenever the value of the difference in ratio between the unstable base line of step (j) and said test ratio curve of step (n) is larger than the value of the difference in the ratio of said unstable base line of step (j) and the ratio of said control experimental curve of step (1) and the differences are of the same sign.

8. The method of claim 7, further comprising quantitating the efficiency of said agent to stabilize said protein formulation; said quantitating comprising dividing, for any given temperature, the difference of the ratio of said control experimental curve and the ratio of said test ratio curve, by the difference of the ratio of the unstable base line and the ratio of the stable base line to yield an estimate of the fraction protein stabilized.

9. An apparatus for determining a state of a cell culture or protein formulation, comprising:

light emitting means for emitting a beam of light at a desired wavelength of light;

means for focusing said beam onto said cell culture or protein formulation;

means for scanning said beam in two dimensions across said cell culture or protein formulation;

means for changing ambient temperature of the said cell culture or protein formulation;

means for calculating the transmitted light through the said cell culture or protein formulation;

means for calculating backscattered excitation light from said cell culture or protein formulation; and

means for calculating an intensity ratio of two wavelengths of back-scattered fluorescent light from said sample, said ratio providing information indicative of said state of said cell culture or protein formulation.

10. An apparatus as claimed in claim 9, wherein said light emitting means is a laser.

11. An apparatus as claimed in claim 10, wherein said desired wavelength is between 500 nm and 700 nm.

12. An apparatus as claimed in claim 9, wherein said ratio is calculated using the following condition (1):

where Λ₀ is the absolute fluorescence intensity of a folded protein, Λ₁ is the absolute fluorescence intensity of a fully unfolded protein, and Λ is the measured fluorescence intensity of a mixture of the folded and unfolded forms at wavelengths λ₁ and λ₂.

13. An apparatus as claimed in claim 9, further comprising a Fourier photomultiplier tube disposed so as to measure said back-scattered fluorescent light and a proportional photomultiplier so as to measure said transmitted light through said cell culture or protein formulation.

14. An apparatus as claimed in claim 9, further comprising a Fourier photo multiplier to be disposed so as to measure simultaneously both back scattered fluorescent light and backscattered incident excitation light from said cell culture or protein formulation.