CA1201990A - Modified proteins and process for the production thereof - Google Patents

Abstract

ABSTRACT

Disclosed is a modified enzyme like protein and a process for the production thereof. In one embodiment, a native protein is grossly denatured. Subsequently, the grossly denatured protein is partially renatured to form a partially denatured protein. The partially denatured protein is contacted with an inhibitor of a model enzyme whose biological catalytic activity is to be modeled. The partially denatured native protein in the protein-inhibitor complex is cross-linked to produce a new enzyme like modified protein. The enzyme like modified protein shows the enzymatic catalytic behavior characteristics of the model enzyme.

Description

~2~19~

This invention relates to modified enzyme-like proteins and -to a process for -the production thereof.
Proteins are biologically synthesized macromolecules having various role in living systems. En~ymes are particu-lar varieties of biologically-active proteins which catalyze specific reactions. Presently, enzyme technology is used in many areas in industry and research such as, for example, medical research, food processing and preservation, the production of fermented beverages, the production of pharma-ceuticals and the anal~tical determination of the concentra-tion of various metabolites and food components by analytical enzyme techniques.
Enzymes are highly specific in their biological activity and generally catalyze a particular reaction at a very high rate compared ~o the corresponding reaction occurring at room temperature without biological catalysis. One enzyme may show catalytic activity with respect to a number of substrates upon which it can act~ Accordingly, a given en-zyme may catalyze the synthesis or degradation of more than one substrate. Some proteins which are not considered classical enzymes, such as bovine serum albumin, show very limited catalytic activity with respect to one or more sub-strates~
Many en2ymes are found in nature in very small quanti-ties. Accordingly, their isolation, purifica-tion and use i5 limited to a small scale operation in view of the expense and time needed to isolate them in a useful form.
Some enzymes occur in nature in relatively large quan-ti ties and are relatively easy to isolate, purify and use. Un-fortunatel~, due to the precise catalytic behavior of the ~. j~,, `~`~

9~0 15~06 enzyme-~ the ~nzyme.~ avail~ble irl l~rge quan~ ieS can only catalyze certain select reac~ion~
Much efor~ has been direc'ced r~ecently ~oward the ~ynth~is of synthetic biolo~i~al catalys~q whis~:h exhibit enzymat~c 5 behavior ~imilar to th~ enzyma~ic behavior e~hibited by native enzymes ~hich are either scarce or exp~n~ive to i~olate.
Further, som~ attempt~ have been ~ade to mc~dify native enzyme~
to change 'cheir enzyma'cic sp~ificity ~o that they m~y ~Eunction to catalyze a rea~cion which they previously collld Dolt ca~alyze.

t)ne technique known to achieve enzyme behavlor to ~atalyze a specific de~ired rea~tion i5 the ~ynthesis of so called ~næyme model molecule~. For example, low ~olecular w~ight compound~
15 may be covalently bonded to functiorlal ~roup~ which exhibit t.le activi~y of ~he ac~ive si~e of an enzyme. ~xamples of su~h preparation~ are descr~bed ln the publications: E~re~low~, R., _vance~ in Chemi~ltry ~eries, R. F~ Grould,Ed., American Che~nical Society, Wa~hingtonl Do C~ 21-4~ ~1971 ) and Tans~, C,, 20 C~; Davalian, D~; ~laungl, P. and E~rs~low, ~ . A_ C'e-.
Soc. , 100, 3918 (1978) an~ Bre~lowf R. 9 I)oherty, JD ~ GUi.1101'C, Go and Lipsey, Cc,, ~_5h~ 100, 3227 ~197S).
Another techn~aue involves the use o~ a synthetic polymer ma'crix whlch i~ ~odi~Eied along it~ baekbone to provide 25 ~unc~ional groups which ~xhibit tlle funstion o~ the ~ ve ~ite of a given enzyme. ~xample~ oi~ such techni~ues can be found in the follo~ing articles- Wulff, G. and Schulza, I., ~srael J.
Chem , 17~ 291 (1978) and Suh, J.. and Rlot~, I. M., ~ ~, 165 (1977).
30 . Another technique involv~s the attaehment of a new chemical

2 --~ . .

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moiety to a native enzy~ne n~ar ~he ac~ive site ~f th~ enæyme to attempt to cau~e ~uch en2y~e to react with a different catalytic activity~ One examl?le of this :Ls the conver~ n of papai~, a proteolytic enzyme to an oxidase type enzyme by the covalent 5 attachment of a 1a~in near the acîiv~ site of the native papain enzyme, as illustrated in the ar~icles: Levine, ~. L. and Raiser, E. T., ~_5~Ce~, 10û ~ 7670 (1978~ aiser, Eo T., et al, ~ ~, No. 191, Biomimetic Chemistry, page 35, l9B0; and Otsuki, To ~ ~akagawa, Y. and ~aiser, E. T., ~ ~, 11,457 ~1978). Other example~ of such enzymati~ l20dif ication ~ay be found in the article: Wilson, M.E:. and White~ide~ G. ~.
S~c., 100, 306 ~1978).
S'cil~ anoth@r attempt to change enzyme æpecifici~y is the in~nobili2ation of a native enzyme into a gel rnatrix. For exa~nple, trypsirl en~yme has been ~mmob;lized in pol5~acrylamide ~el. The polyacrylamide gel allows amino aeid esters ~o diffu~e through the gel ma rix to rea~ with the erlzyme but wi:Ll not allow larger proteins to diffu~e t~rough. T~uSr the enzyme spec~ficity ~s changed by e i~inatirlg access o~ one o the subs~ra~e mol~cule~ to the enzyme.
~rhe in~obilization of native enzyme~ is well established in the artO Al~o, examples of ~nzyme spec:ifici~y changes by immobilization are known in the artO Both immobilization and enzym~ speci:Eicity chanse~ are described in the ~19e~ Chemical ~, 3 Ed.~ 9, 148 (1980 published by Wiley and Son, Inc.
Two other methods relating to enzyme immobilization are disclo~;ed iD ~lo So Patents 3,802,997 and 3,930,950. ~n U. S.
Patent 3,802~97, a method o stabilizirly enzymes by bonding ~he .~ L9';~6~

enzymes to inorganic carrier~, în the pre~ence of their substrates, ~hereby the en2yme is immobilize~, is disclo ed. In U. S. Patent 3~930,950l a method of enzyme immobilization is di3clo~ed whereiYl an active ~uppor~ member i~ providea which is capable o~ reacting wi~h an enzyme ~o become chemically bonded thereto. ~ub~equently, ~he active suppor~ is contacted with an enzyme~substrate complex which has been formed by mixing to~ether an enzyme and a ~pecific ~ubs'cra e, while minimizing the tran~iEormation of substrat~ to proauct . Thus ~ the enzyme compone~'c of the cosaplex becomes chemically bonded to ~he suppor t member .
Al~o, ~ t ha~ been known that a native ly~ine mono-cxygenase ca~ be reacted to block the sulfhydryl groups on the enzymer When the ~peci:Eic. enzyme ly~ine mono-oxygenase is so treated, it 8how8 new catalytic activity toward amino acid~ and catalyse~
oxidative deaminat~on instead o~ its na~ural oxlrgenative de~arboxylation~ 130s~ev~r, the reporters cannot ac~ount for the modiied behavior~ See the article by ~ramauchi, q~.; Yamamoto, S ~ and ~ayai~hi 9 O o ~ in T ~ ~ ~
24~, 10, 3750-3~52 rl973)~ Al~o) it bas be~n r~ported tl at by reacting a na~ive enzyme, for e~ample trypsin~ w~th its natural ln~ibitor, a~d sub~equently cros-s linking the enz~me, its acti~ity with respect to itg natural subs~rates c:an be ~odifiedO 8ee l.h~ ~r icl~ by Beaven, G. ~. and t~ra~zer~ W. B
~n ~ ~e~Re-,, 51~ 215-18 (1973)o Al~o; synthetic prot;~ins have been syathesi~ed by 'che . an~horing of an amino a~d residue on a ~:olid support and subse~u~ntly adding amino acid residues one after another.-- Further~ semisynthetic proteins have been syn~hesized by a 3û method wherein a native protein is ~ubjec~ed to limited /

hydrolysis to produce protein fragments. The fragments of the native protein are then subjected to a process whereby one or more amino acid residues are added or removed from the frag-ments to form modified fragments. The resul-tant modified frag-ments are then reattached to form the semisynthetic protein wi-th an altered amino acid residue composition. E~amples of the synthetic and semisynthetic protein technologies cited immediately above are found in the book Semisynthetic Proteins by R.E. Offord, published by John Wiley and Sons ~td., copy-righted in 1980.
While these techniques are sui-table for many applications, a need exists for a simple, efficient, economical and systema-tic method for chemically modifying an inexpensive and commer-cially available native protein to produce an enzyme-like modified protein. The protein can show a catalytic enzymatic activity with respect to a desired chemical reaction which was not previously a commercially-useful reaction catalyzed by the native enzyme and which new reaction can be predetermined in a systematic fashion. The methods disclosed in the above-disclosed references simply subject an enzyme to a set of con-ditions and attempt to eludicate its behavior. They fail to provide a systematic method to modify protein characteristics.
The present invention achieves an enzyme-like modified protein by convertlng a naturally occurring so-called na-tive protein to an e~zyme-like modified protein exhibiting differ-ent characteristics than the native protein starting material.
Accordingly, one aspect of the invention provides a pro-cess for chemically altering the substrate specifici-ty of a na-tive protein to produce an enzyme-like modified protein, com-prising selecting an enzymeto be mode]ed, grossly denaturing a nati~e protein, partially renaturing the grossly denatured native protein to produce a partially denatured pro-tein, contacting the partially denatured protein with an inhibitor for the model enzyme to form a partially denatured protein-model en-zyme inhibitor complex, and cross-linking the partially de-natured protein in the protein-inhibitor complex.
Another aspect of the invention provides a process for chemically altering the substrate specifici-ty oE a native protein to prod~ce an enzyme like modified protein, com-prising, selecting an enzyme to be modeled, grossly denatur-ing a native protein, admixing the grossly denatured protein with an inhibitor o~ the model enzyme, partially renaturing the grossly denatured protein, in the presence of the inhi-bitor, -to produce a partiallv denatured protein inhibitor-complex, and cross-linking the partially denatured protein portion of the protein-inhibitor complex.
In one embodiment of the invention, a native protein is grossly denatured by contacting the native protein with a - 5a -denaturing agent, for a time and at a ooncerltration sufficient to cau~e e~entially gross protein denaturation. Next, ~che gr ~ ly dena'cured protein is ~ubjected to ~onditiorls that cause the partial renaturation of the protein to produce a partially 5 den~tured native pro~ein. The partially denatured protein 1~
contacted wlth an inhibitor of a model enzyme, whose catalytic activity i~ to be mo~eled, to ~orm a partially denatured protein-inhibitor comple3cO The partially ~natur~d prote~s~ in the complex i~ next c~o~-llnked tcs produ~e a new enzym~ like 10 modified protein.
Sub~equently, the ~odel en2yme inhibitor and any exce~s cross-linking ~gerl~ are removed fEOIII the n~wly formed enzyme- -l~ke ~nodified proteln to yi~ld a unctional~ stable, analogue ~co the model enzyme. The enz~me-like modified protein thu~1y 15 produ~ed exhibi'c~ the catalytic activ~ty characteristies of the ~sodel enzyme.

~n attaining the advanta~e~ of the present invention,, i~ has 20 be~n di~co~ere~ th~t a prot~in can be modified from it~ native conformation 'co a modified conformation by practicing the pro~ess of th~ pre~ent in~ention. The i~ew conformatlonal s"cate ~f~nes an ~nzym~-like modlfied p~otein.
A~ u ed her~inr the ~ord ~nzyme~ i~ de~lned a~ a protein 25 whlch ha~ w~ known catalyt~ c actl~ty 'coward spe~i~lc substrat~. The ter~ ~prolt~inM a~ used herein is de~f ined a~ -. generally accept~d i~ lthe art~ to wit, a poly~eptide for~ed o ~mino ac:id~ 'co yi~ld ~ biological molecule.
Th~ pr~ce8-R s:~f 'ch~2 present in~rention comprises chemically 30 loodifying ~ native protelrl from one conormation, its na~ural or

3~

15~6 na~ive state, to a ~econd conformation, a new modii~ied state.
The proce~ produces a new, enzyme-l~ke Tnodified pro'cein which is produ~ed to yield a stable, new en2yme-like modifiedl protein which ~odels one or more of the en%ymatic ac~ivi y 5 characteristics of the selec~ed model enzyme.
In the preferred embodiment of the inventiorl, a na~ive protein is selee~ed which ~ to be chemically modified to produce the new enzyme-like modifi~d pro~ein analogue of a de~ired model enzyme. ~he process of the present invention 1~ convert~ 'che ~oluble native protein, which does not pos~ess the desired catalytic ac~ivity, namel~ the enzymatic ~atalysis behavior c~f the model enzyme, into a stable, enzyme-lilce modified protein which mimics or copies the biological catalytic activity characteristic~ of th~ moael enzyme.
~ pre~erred way of carryillg ou~ the novel proce~s o~ ~e pre~ent invention or chemically modifying a na'cive ~rotein to produ~e a predeter~lined es~ ne like modifiea protein cor~pri~es the step~ of: grossly denaturing the native protein by ~ontac~.ing the native protein with a denaturing agent~ or a 20 ~ime an~ a~ a concentration ~uffici2nt to grossly dena~ure the native protein; partial~y renaturing the gro~ly denatured protein ~o produce an only partially dena u--d prot~in by contacting the grossly denaturea protein with reagents or process condi~ions suf~cient to par~ially rena~ure or refold 25 the prote~n; contacting the re~ultant only partially denatured . protPin wi~h an inhibi~or for a selacted model enzyme, for a ti~e ~uf~icient ana a temperature sufficient, to produee a partially dena~ured protein-inhibitor complex and subsequently cross-linkiny the partially denatured protein in the complex to 3~ orm the new, stable ~nzyme-like modified pro~ein by contacting 7 _ 19~

15~0~
th~ parltially denatured protein with a cros~-linkirlg agent. Any exces~ cross-linkiny agent and l:he model enzyme inhibitor are removed fro~ the newly created ~nzyme-lik~ modii~ied protein to isolate the new, catalytically clctive enzyme~like modified 5 protein produc 1: O
Wh;le in the pre~erred embodliment, a nonen2ymatic na'cive protein s'car~ing materials or so-called hos~ protein i~
converted lnto a catalytically acl:iv~r enzyme-like modified protein, the ~onver~ion o~ any native pr~tein to an enzyme-like 10 modified protein analogue of a model enzy3ne is co2ltempla~ed herein. ~;en~rally~ na~ive or host pro'cein~ which are mos~
readily available :Eor conversion in'co an enzyme~like modi~ied f~rm are nonenzyrQatic prol:eirl~, like bovine ~erum albu~in.
Preferably, the Y~ative protein starting material i~ selected 15 ~ecauæe it is availabl~3 in rea~onably pure ~orm, in commercially useful qu~ntitie~, at reasonable unit co~tæ. ~owever, 2æ
exemplifi~d hereinafter, any protein can be used ~s the na~ive protein s arting material, either enzymati~ or nonenzy~aatic proteins. Typic~lly nonenzyTna'cic proteln~, like bovine serum 20 albumin, are availabl~ in pure formO in large quan~iLti~ at lower co~ han enzymatic prote$n~, and are thll~ preferred ~tar~ing m~aterials.
As disclosed above, the ~tarting ma'cerial native protein i8 yro~ly deJ-atured or un~olded to essentially de~troy its na~ive 25 t~ree dim~n~ional che~ical stru~ture. Next, to ashieve a pro~ein structure whic~ is capable of binding inhibi~or 9 although not the na~ive ~tructur2, 'che grossly denatured proteln i~ partially r~folded to produce a partially denatured protein., ~he par'cially d~natured protein has neither the ~:hemical ~0 structure, so called con~ormationr of its parent, the native 9.~3~

15~06 protein star'cing material, or o~ the gro~3sly denatured protein it was produced immediately- ~ron~. While no~ b~ing bound by any theory, it ii3 belie~red ~hat the gro~s denatura~ion of the natlve pro~ein ~arting ma~erial and then only partial refolding thereof allow for potential ~ew $nhibitor binding ~ites to be generat~d. Thus, different sites are generated ~han would be available if the native protein itself were par~ially denatured with~ut going through a gro~s denaturation step prior to contacting the protein with ~he ~nhibitorO
As use~ berein, the phrase "grossly dena~ured" is d~fined as generally accepted in ~he art, to wit~ a major change fro~ the native ~tate which causes the proteirJ to l~ecome essentially ~ompletely un~olded~, In the grossly denatured state, the protein lack~ both secondary and ~rtiary ~tructure and 15 re~emble~ a ran~om co~ l . The gro~sly denatured state can be determined by co~pari on of phy-~ical mea~uremen~s o. ~he gros~ly d~natured protein to those of a iaod~l random coil. ~xalaples of such phy~ical ~a~urements i~or 'che determination of gros~
denatur~tion ~re ~7isco~i'cy, circular dichroism~ sedimentation 20 coe~ficien~ and ultr~iole~ ~pectraO Withi~ the d~fini~ic~ll of ~ros~ly denatur~d protein~ are 30m~ pr~3tein which ~ay contain a tra~e of residual -~econdary ~nd/or tertiary structure~ Such tr~ce~ of r~dual 3'cructure do XlOt ad~rersely e~Pe~t he operat~on o~ the pre~ent invention.
~5 Tha~ characterist~e~ of gro~sly denatured prot~ins are ~ell-kno~r~a in th~ art., For @x~mple, when using a visco~i~cy measure~ent to Dtonitor prot~in ~naturation, the v'isc08ity oi~ a globular pr~teizl generally gues up during the unfolding proc~s. On th~ other ha~dO ~he viscosity of fibrous protein is 30 quite large in the native state and may either increase or decrease upon unfolding. Such mea~urement changes during denaturation are well li~ted in the~ erature as described hereinafter. ~n~olding can also be monitored by th~ change in ultraviolet spectra. The major change in spectra ~ccurs because s of the transfer of phenylalanyl, trypyl~ a~d tryptophyl side chains from the protein interior ~o ~Ae solvent environment durin~ dena~uration. Generally ~he mol~r absorptivi~y will decrease at some wavelength~ while increasing at other~ due to the 3hi~t in absorption bands. The grvss denaturatlon o protein ~tructure dramatically changes the optical rotation and c~rcular dichroism spectraO The exact nature of the changes relate to the secondary and tertiary structure of the na~ive protein. These physical parameters, used ~o determine gros3 denaturation, as well as many other~, can be ~onitored to . determine when a protein i~ grossly de~atured a~ des~ribed ~n many papers on protein denaturation such a~o Charles ~anford, Ad ~ 23, 121 (1968).
Several review article~ des~ri~e the pr w es~ of ~ross denaturation and the criteria for de~onstr~tlng gross denaturativn in ~erm~ generally accepted in tha art and u~ed accoraing to this i~vention, namely, the following arti~les, Charles Tanford, Adv. Protein ~ , 23t 121 (1968~; P.
Privalov, Adv~ Protein ~ , 33, 167 (1979); and C. ~.
~n~insen, Science! 181, 22~ (1973).

o~

1~4~6 The preferred ~ethods of protein gro~s denaturation accordi~g ~o the i~ven~ion are as follows.
Gros3 denatura~ion of a nat~.ve protein starting material can be achieved for ~o~t pro~ein~ ~y the use of guanidine byarochloride (~u~Cl~o Gro3s proteln den~tur~tion is usually ~omplete by concen~rations of r~m 6 to 8 ~ GU~Clo In many instances, urea can be sub~ituted ~or Gu~Cl in a con~entration of about 8 M to achieve qro~s dena~uration. Gross denaturation can al~o be achieved by rai~ng the temperature until the well recognized un~olding transition takes place. ~oreover extremes of acidi~ or ba~ie ~ondition~ alone or in combination with te~perature can be used. Generally~ for most proteins a temperature on the order of 50-70C. is sufficient t~ grossl~
denature t~e native protein ~tartiny ~at~rial~ Suit~ble acids for gros8 ~e~aturation include acetic7 formic, propio~ic and citric~ ~hile ~uitable ba~es include ~odiu~ and potas~ium ~ydro~lde. ~ui~abl~ inorganic a ids as dena uring agents in~lud~ nitric, pho3phor~c, sulfuric and hydrochloric.
~hen ~he ~ro~ d~naturi~g condition~ are reduced the protein ~olecule~ beco~e~ p~rti~lly renatured. Thi~ partially renatured state ~ay con~ist of pro~e~ molecules that remaln nearly ~otally d~natured mixed ~ith other molecules are nearly completely refoldedO ~n the alternative, nearly all protein ~olecule~ are ~arly compl~ely refolded. In the ~l~erna~ive, ~5 ~arly ~11 proteln molecule~ could be partially and ne~rly ~qu~lly refoldad~ ~he e~act physical state of the p~rtially g~

renatured protein is not critical to the present inven-tion.
The partially renatured protein state will be recognized by the changes in physical parameters which indica-te that the protein is no longer a random coil but contains considerable secondary and tertiary structure. The partially renatured state, however, ~acks the highly ordered s-tru~ture of the na-tive protein state and is believed to afford different poten-tial sites for inhibitor binding than a partially denatured native protein. The partially renatured protein is believed to offer different binding sites for the inhibitor since it has been completely structurally rearranged due to the gross denaturation step. The grossly denatured protein can be partially renatured to offer new sites, not previously offer-ed in a partially denatured native protein, for inhibitor binding.
The partial renaturation of grossly denatured proteins is well known in the art and discussed in detail in the fol-lowing references.
L.G. Chavez, Jr. and H.A. Scheraga, Biochemistry, ~1980), 19, 996-1004; H. Taniuchi and C.B. Anfinsin (196~), J. Biol.
Ch _o~ 243, 4778; ~. Taniuchi and C.B. Anfinsill (1969), J.
BiolO Chem., 244, 3864; M~Io Kanehisa and T.Y. Tsong, Biopolymers, 18, 2913 - 2928 (1979); EoW~ Miles, Ko Yutani, and Ko Ogasachara, Biochemistry, 21, 11, (1982~ and D.B.
Witlanfer, Adv. Protein Chemistr~, 34, 61 (1981).
After the protein is par-tially rena-tured it is contacted with an inhihitor of a model enzyme whose catalytic activity is to be mimicked.
As us~d herein, the term "inhibitor" means any compound with sufficient structural similarity -to the natural substrate of a model enzyme to serve as a template for the catalytic site of the enxyme~like modified protein. In the preferred embodiment o~ the preparation of an enzyme-like modified protein, the inhibitor is any of the Icnown cla~ical inhibitors for a gilren model e~zyme. ~owever, aæ used herein ~inhibltorl' 5 can include any molecule with sufficient fitructural similarity to the classical inhibitor to pre~erve an inhibitor like site on the modlfied protein. The natural substrate of . he model enzyme can act a~ the ~nhibitor or template for the modified protein in many cases~ Inhibi'cors are generally not degraded by the 10 en2yme~ as ar~ ~ubstra~e~, and ~erve ~o more readily preserve a catalytic ~ite than the na'cural substrate. One example of the ~ructural ~lmilarity of an enzyrae inhibitQr and the natural ~ub3t~ate of an enzyme i3 the case of gluco~e o~cidase. Glucose i~ the natural su~3tra~e of gluco~ oxidase while D glucal iæ
15 the inhibitor' fs~r ylucos~ osidase~ Gluco~e and D-glucal are very Btructurally similar,.
In the preferre~l embo~iment, after the partially rena~ured pro'ce$ri ha be~n contact~d with the inhibitor for a time ~ufficient ~nd at a temperature ~uf~icient to form the protein-20 inhibitor coDple~ the parti~ renatured protein por~ion of theco~ple~ i3 cros~ 2ked to s~abili~e the n~w ~tructure.
As u~d herein, the ter~ ~ro~s-linking~ means the formation o~ covalenlt bond~ betwe~n r~ctive ~ites on a protein.
~en~rallyr pro1:~in ro~-linking is acco~ ed by the use o~
25 raultifuri6:tlorlal re~gents u~:h i~3 glutaraldehyde. O~ber examples o~ ~u~table cros~-linking reagerlts to eiEfect the cro~s-linking of a protein are: 2-amino-4~ 6-di~hloro-~-triazine; dîazonium ~alts; ~-bydroxysuccin~mide; p~ e~zoylazide a:nd ~ho~e reagents di clo~ed ill the followin~ reference~,. WoldQ ~., Method6 30 ~, 11, ~dited by C. ~,. W. ~irs, C.~.~ir., Academic Press~

~o~9~3~

1967, 617; ~asold~ El. et al., ~ _ _, 10, 79~ , 197 and ~eyes~ M. Bc, in th~
Chemical Technolo~ ~ 9, 3rd Ed,~, 1980, J. Wiley & Son~, ~nc~, 148-1720 Cros~-linkirlg may also ~e acbieqed by di~ulphide 5 rearrangement~
One pre~erred process to grclssly denature a native protein ~tarting material wi'ch be~a-mercapl:oethanol i~ the ollowing.
Th~ na'ci~re protein is admia~ed with beta-mercaptoe'chanol in a molar ratio of about 1000 unit~ of beta-merca~toethanol to one 1~ unit of protein. After ~reatment with beta-mercaptoethanol, at neu~ral pR, for one to geveral hours, quanidine hydro~hloric is added to a concelltration of 6 1~. Th~e conditions are sufficient to totally denat~re most pro ein~. Preferably, the concen'cration of protein and p8 æhould be such that part or ~Do~t 15 of tha protein remains in ~olution- The be~a~mercaptoetharlol cleaves di~ulphide brid-3e~ and facilit~e~ the gros~.
denaturat~on of the protein by as~isting in ~tru-::ture degradation.
In the preferred embodiment of the ~nvention, a nat;ive 20 prot~in or host ~protei~a ~ho~in~ little or no catalytic activity wi~h re~pect to a selected ~ub trate is converted chemically by the process of l~hQ present inv~ntion into an enzyme like oodified pro~ein analogue of a mod~l enzyme. Many enzymes are 3us~eptible to modeling or mimicking by the pre~ent proces~ to 25 produce their eazyme-like modified protein analoyue~ ~rom Rel~ ed na~ive proteirl starting n~aterials. Example~ of such model enzymes whi::h are su~ject to enzyme-like modi~ied protein analogue production are hydroly~ic enzymes, redox enzyme~ and tr-ansferase enz~nes~ y way of example: ~he first group, 30 hydrolytic enzymes include proteolytic enzymes which hydrolyze ~ 14 --r ~

15~06 protein~r e.g., papain, ficirl, pepsin, trypsin, chymotrypsin~
bromelinJ keratina~; carbohydrases which hydrolyze carbohydrate~ eOg. " c:ellulase~ amylase, maltase, pectinase, c:hitana~e; est~ra~es which hydrolyze esters" e.g., lipase, 5 choline~teraæe~ le~ithinase, alkaiine and acid phosphateases;
nucleases which hydrolyze nucle;c acid, e. g~ t ribonuclease, ~1eoxyribonuclease; and amida~es which hydrolyze amir.esl, e.g. ~
ar~inase~ ~sparagina~e, glutinaE;e, histidase, and urease. The ~econd group are redox enzymes that catalyze oxidation or 10 ~eduction reaetions. These include ~lucose oa:idase, xanthine oxidase, catalase~ peroxldase, lipo:~idase, and c~rtochrome reductase. ~rl the third group are transferase enzymes that l~ranser groups ~rom one lQolecule to another. I~:xamples o~ these are glutamicpyruvic transaminase, glutam~coxalace~ic 15 transam~na~;e, trans~ethylase, phosphopyruvic tran~phosphoryla~3~ 0 In th~ u~ual pr~cti~e of the E~resent invent~on, one s~lects a flrst or ~odel er zy~e. One then sel~ct:s a ~econd na~ive or so-~:alled host protein to ~e modeled after the model enzyme ~o 20 produs:e an en2y~@-lik~ ~odified prol~ein. As di$cu~sed above, in mtany ca~@~ the native ~rot~n is itsel~ enzyma~ically ~ctive, w~'ch resp~c~ ~co ~ given sub~trate~, since many common enzy~es ar~
av~ilabl~ in 3.alrge ~uantitl~ t ~airly low co~ts i n homogeneous ~ple for~ owever, nonens~yma~ic protein~ ar2 egually use~ul 25 ~hen th~y c:an be Qither purc~a^~ed in pure ~or~ or purified by ~:onven~ional m~ans ~or u~ wi~h the pr~sent proc~sO One exaD~ple of uch a nonen~yma'cic protein which may be used as a na~i~re p~otein for the starting material of the bovine s~rum albumin (BSA~,. BSA is availabl~ in relatively pure form at 30 iEairly lo~ co~t from numerous commercial ~ource~.

~ 15 --By practicing the process of the pr~sellt invention, one can t:ustom-tailor the native protein to a diferent stable enzyme-like modified protein form which shows the catalytic a~tivity charact@ris~ics of 'che enzyme wXich ha~ been mod*led. The 5 ability to cu~tom-tailor a native protein into a predetermined ca~alyti~ acti~ity provides greater a~vantage~: iss a wide range of chemical and in~ustrial si'cuations~ For example,, if one ~ishes to use an eDzyme which is in short supply, i8 very ~pen~ive or ~ery diffi~ult to isolate and/or purifyv such an 10 enzyme may serve as a model ~nzyme for the preparation of an enzyme-like modified protein analogue by the pre~ent process ~o mimic its activi~y.
Thus, a native prot2in which is availabl~ ~n large quantities or at low co t ~an b~ reformed or ~Qodified by 'che 15 proce~ o~ the pre~ent invention to convert the avail~ble native prol:ein ~tarting material into an enzyme-lik~ r~odified protein forr~ of a leææ available andfor more e~pen$ive erlzyme.
In the preiEerred eml;~odiment of the invention, a nativ*
protein ~arting ~naterial i~ purified and dissolved in a near 20 neutral aqueou~ solvent in the pre~ence o~ a suitable buf~er to maintain the solution near neutrality. Subsequer~tly, the ~ativ~
protei~ is ~ro~sly dena~ured by any of the expedients de~c:ribed hereinabove to produee a ~1:0~8ly denatured, preferably, 801uble ~rm of th~ native protein starting material. Next~ the grossly 25 denatured prote:in is par i~lly reoatured or rePolded to produce a partially a~naturedl protein., Sub6equen~1yt an inhibitor for . the model ~nzyme i~ admi~ed with the partially denatured prs~tein. Suficien'c time and sufficient temperature are provided for a partially aenatured protein-inhibitor complex to 30 form. Subsequently, to preserve th~ new, en~yme~like modified t. ~ !3~3 15~06 proteinl, the partially denatur~d . nat~ v~ protein por'cion of the protein-inhlbitor complex mus'c be stabilized~
The new protein i~ ~tabili~ed by cros~-linking o~ the protein to produce the enzyme~like T~lodified protein. Often, 5 cros~-linking i~ done as d~sclosed albove by glutaraldehyde cross-linking agent ~lnce ~t i~ ~ne~pen~ive. However, any o the above-de~cribed cros~-linking agent~ can ~e utilized effectively ln the ~onveYItis~nal manner.
The proce~ o~ the pre~ent invention 1produces a new, enzyme-10 like modificd pro'cein which eachibit~ a number o~ advantages allduses~, By the di~coveries of the pre~ent invention an enzyme-like modified~ protein can be produced which i5 stable and e~hibits a new enzyme-like cataly'cic activity which w~s not presen'c in the native proteill. Su~h modl~ied prot~in~ ~howing 15 enzyme -l$ke catalytic behav~ or are u~ePul to p~rform catalytic anabol~c and ~at~bolic reaction~ in~tead of a na'curally occurrin~ enæy~eO
~ :n ~11 embod~laerit~ of the pre~enlc invention th~ inhibi~or of khe mod~l enzyme 1~ removed after gynth~si~ of th~ enzyme-like 2~ mod~ f~ed proteinO T~ Ga11Y repeat~d ~ashings of the oblliz~d mod~fie~ protein ~ ~uffic~ent to remove the lnhibitor. Buffee~d asaueou~ ~olu. ion ~an also b~ u ed to remove ~h~ l~hibl~or ~, ~uch ~uffer~ ar~ ~xemplified here$nafter~
Other eloboai~ents of th~ present invention will be apparent 2~- to thos~ of ordinal.y ~kill in the art from a considerativn of thi~ ~pecification or practice of invention di~clo~ed herein.
int~nded t:hat 'che Bxample~ in the specif i~ation be ' '~ ' ' 9~3~

~on~id~red as exemplary only w~th the ~cope and splrlt of the ~nvention b~ing indicated by the claims~ The ollowing Example~
are exemplary of the var~ou~ emboaiments of the process of the present invention di~cu~sed hereinabove~
~XAMPL~ 1 Sixty mg of chro~atographi~ally puriied pancreatic r~bonuclease ~RNa~e1 ~ative ~nzyme, pur~ha~ed from Slgma Chemical Company~ lot llOF-C2051, ~pe I~A, ~o. R-~000, i~
dissolved in 3 ml,of 8M ur~ denaturing agent containing ~1 M 2-amino-2-~hydroxymethyl)~1,3 propanediol-~Cl buffer (here~n~fter tri~ buffer)~ a~ p~ 8 with ~lo~ stirring ~t 25bC.
Next~ 0~1 ml o neat beta-~ercaptoethanol denaturing a~ent 18 added in the ~olut~on to break ~isulphide bond~. ~he re~ulting ~olution i~ shaken slo~ly for 15 minute~ at 25C~ Th~
~olut~vn i~ then placed in a ~toppered erlenmeyer fla~h under a nitrogen at~o~phere for 16 hours at 25C. The clo~ed environment of th~ fla~k is ~intained by a pressur~zed nitrogen tank releasing nitrogen ga~ into the flask w~h ~he flask vented lnto a water trap.
Ne~t, 3 ~1 of the solu~ion i~ ~hromatographed acros~ a g~l filtration ~olu~nr whi~h is 12 in~ x 1 in~, containlng Sephadex*
br~nd G-15 gel ~ ration materialO Sephadex is a cross-llnked~
- bead~d, high ~ol~cular ~ight polysacoharide which ha~ been cross-linked with epichlorohydrin~ marketed by Pharmacia ~ine ~5 Chemical~. Ace~ic acid at 0.1 ~ pH 3, 1~ used as the eluan~ for the 9~1 column. The eluant flow r~te i3 maintained at 1 ml/min ~ia a ~low speed perl~talic pump. An absorbence ~onitor i~
placed at the outflow point of th~ chromatography column and an ultravlolet ~etector ~et at 276 nm ~s u~ed ~o detect eluting protein fractlon~. A ~ajor protein fraction of about 5 ml *trademark o 1~0~
elute~ from ~he column under thes~ conditions and ls colleeted in ~ $~all beaker. Th~ pro~ein raction i~ analyzed u~ng an ACTA III*spectophotometer fro~ Beckman In~trument3 Company. The molarity of the protein raction i~ det~rmined to be 2~67 x 10-5 wherein the 280 angstrom absorbence coefflcient ~5 0.254, the ~olecular w~i~ht o the protein i~ ]L3000 daltons and ~he extinctlon coèfflcient value, Ed is 7~3 for a 1% solution.
To the 5 ml of the protein c~n~ainlng eluant collected above i5 addea 1 ~1 of loO M tr~ Cl buffer, p~ 8; 1 ml o 1 mM
~thylenediaminetetr~aGet~c acid ~EDTA); 1 ml of 5 x 10-4 M
reduced glutath~one; 1 ml of 5 ~ 10-4 M of o~ldized gl~tathione and 1 ~1 o 0.02 M trypta~lne-~Cl inhlbitor. The tryptamine-~Cl 1~ a~ded a~ the inhibi~or, while the other reagents tna~ely, the ~DTA an~ glut~thione) are added in accordance ~ith the tea~hin~ of Lloyd Chavez, Jr~ and ~arold Scheraga ~n ~Folding s Ribonuclea~e, 5-Protein and Des (121-12~ ribo~uclea~e ~ur~ng Glutath~one Oxldation of ~educ~d Prot~in~, 1980, ~s~ gL~ 19, 996-1004. The resultant solution ~how~ a p~ of 5.5~ Th~ ~olution i~ ~tirred for 1.5 20 hour~ ~t th~ 5.5 pk under ~low stirrlns. 5ubsequently, the p~
Eaised to about 8~0 by ~h~ addition of 100 mg of tris base ~ry~tal~ The re~ulting tri~ ~ontalnlng solution is stirred slowly ~t p~ 8, at 25C~ ~or an a~dition~l 2.5 hoursO ~ fluffy ~hite pr~ip~tat~ 1~ for~ed during the ~tirring pro~e~ t the end of the 2.5 hour p~riod~ the ~olution i5 dialyzed again~ 1 trls ~Cl bu~, p~ 7~2~ containing 0~005 M rypta~ine lnhibitor. The dialy~is me~brane i~ a Sp~ctrapor*brand membrane ~ubing~ ~o. 2 ~i~e, hav~ng ~ ~olecular ~eight exclusion range of 12-14,000 dal ons. The dialy~is procedure includ~d two buffer ~hange~ ~th a gro-~ volum~ of 7000 ml of buffer over a 17 hour period, at 0.5~C, with 810w ~.haklng o~ the dialysis tubing ln ~he di~lysi~ ~edia.
At~ dialysi~r 2 ml o the ~nzyme-like ~odified protein ~, .
1~ --lS~06 c:ontain~ng ~ lution ~ hron~ate~graph~ea ~cro~s a Sephadex*brarld G-15 column, the columrl dimen~lon beinly 1~2 ~n~h x 12 inche the column be~ng illed ~ith gel iltr~tion agent u~ing 1 m~3 ltri~
~Cl buiEfer at p~ 7 a~ the eluant. Such a gel ill~ration column i8 used to ~eparate low il~olec:ular wei~ht material~ from proteis material~. Th~ eluant fîow rate i~ 1 ml~'min. The eluant wa~
monitored a'c 254 nm to detect elutirlg en~yme-like modified proteln. A fir~c porl~ion of elu~lng ~nz~sle-like modified peo~ein i8 coll~c~a*d an~l as~ayed V8. ~ubstrate for e t~rase enzyme. The substr~te used ls tr~p ophan methyl ~ter (TME~ e the sub6trate being purcha~ed from Slgma Chemical Company, lot lO9C-0048, No. T-5505.
The ac'civi'cy of th~ ~stera~e enzyme-like modified protein t2sted ~ ~ollow~. The protein fra~t~ on ~ollected abo~e i~
a~sayed by high ps~ure liqui~3 chromatography (hlPLC) to determ~rae act:lvity toward 'che TME ~ulb~trate. The a say conaiti~:~ns are as follo~,. The ~PLC co~umn eluant 13 0003 M
ac~ate~ p~ 6. The colu~n ~upport material is porou~ 8iliCZI
bonded phase materi~l ~ontaining ~arbo$yl ~ide ct aln~9 The column is 8tainl29~ st~el composition and is 2 rDm x 25 ~m, with a flow ra~e of 4 ml/~in~. maintainedl by cc~nventional high pres~ur~ pump~ iEor example arl Alt~x* P~odel 110 A pump. Th-2 column eluant i~ d~t~c:ted l:)y a convent~onal ul~raviolet mcanitor at 254 nm~
~rh~ a~say ~ample i prepared a~ follow~. One ml oiE the e~tera~e ~nzy~ e mod~f~ed proteln (with an absorptie~n of 0.222, at 280 nm~ equallng 0.335 mg~ is admixed with 8 ml of 5 mMI tri~-~Cl buffer~ pR 8 and 1 ml of 0.1 ~1 TME substra~e. The ~ssay conl rol ~olution is prepared by adding 8 ml of 5 mM tris-~Cl buffer, pl~ 8r to 1 ml of 0.1 P~ TME and 1 ml of the ~ri~
. *trademarks 3~

blaffer, p~l 7y u~ed as the eluan~c solu~ion. The pH of both the control and ~ay sample soiution is 6 . 6 O
One ml of the sa~ple is removed from the beaker, uslng a 2 ml h~odermic syrirllge" with an 18 gauge needleO Th~ one ml of ~ample is pas~ed throu~h a 20 microliter in~ection ~ample loop onto the! ~PLC ~olumn. The inje~tion time iæ re~c~rded. ~his procedure is repeated using ~he control 801UtiOrlo Both ~ample and con'crol are chromatographed 4 t~me~ in order to obtalFI a plot of mola~ lty of tryptophan versu~ time for ~ample and control to elu1:e from the colun n.
A stati~tical analy~ perEonned with the d a'ca obtained from the abo-re procedure which shows a slo~e of û.3171 x ~û-5 P~/mi~l., ~ith an error of 0"00~85 x 10 ~ ~q/min. is determined $or the ~ample. A slope of 0.2B78 x 1û-5 M/min. wi~h an error of 0,.0091 x 10-5 P~'min. is deterrd~ned for the oontrol.
The following expre~lon 18 used to calculate 'che ac:tivity of the e~ter~e enzyme-lik~ modified protein accor~ing to ~he pre~nt lnventionO
~c:t~rit~

~he~r~$n: ch~nge i~ 810pe iS ~che difference be~ween ~h~
ple a~ ontrol ~lopes (in thi~ example 0~,0293);
~ror i8 the ~um oiE the ~rror~ in the ~ample and eontrol ~lope5 ~in th~s example 00016); Vs is the ~a~aple volum~ ln litersO 10~ is micromoles/mol~; and ~ ng of ~odiie~ protein in th~ as~ay. .r In th~ ~bove c~alcula~iont the concentra'ciorl o~ ~he modified E~Eotein i~ c:alculated ~rcsD ilt~ ab~orbance at 280 nm~ uæing th~
extinetior~ ~:oef~ic~ent fo~ ribonucleaE;e, which is 7..3 for a 1 Qolu~ion. The extinc~ion coefi~icient ~s disclosed in the r~f~E~nce erltitled ~ 5, 49-62 (1973) au'chor~d by D. M. Xirschenbaum.
The a~3ay re~ults area as ollows:
Substrate ~ME ( ~anit~3/g ) Ini'cial ac~ ivity O, 000 Final activity 8.0 ~ 00717 The re~nlts show that the e~ rase enzyme-lik~ ~nodif ied protein prepared according to the preæ~nt invention exhibits activity with respect 'co the esterase enzyme sulbstr~te TME wh~re 10 no activity is previously deJcected in native r ibonuclea~e enzyme~ ~his illustrate~ the conversion o one genu~ of en~yme, ~amely, a nuclease to a ~econd genu~ of protei~, namely~ an e~'cerase enzyme-like modified pro~ein.
The results ~how that the e~terase enzyme-like modifi~d 15 protein prepare~ ac:cording tc~ the pre~ent invention ex~ibi~^s activity with r~spect to the e~ter~se enzyme !3ub~tr~t:e ~M~: where no activit~ is l?reviously detected ia native ribonuclease enzyme. ~hi~ illustrates th~ conversion of one genu~ of en~ym~, namely, a nuclease, to a second g~nus of protein, namely, an 2n est.eras~ enzyme-like modiied protein.

~X~MPL~ 2 -One hundred and twenty mg of chromatographi~ally pur~fied bo~ine pancr~a~ic ribonuclea~e (~ase3 native eQ2yme,pur~hased from S~gma Chemical Company, lot llOF-02051 Type IIA~ ~o. ~-5000, is dis~olved in 6 ml of 8 ~ urea containing 0.1 M ~ris~
~Cl buff~r, p~ 8 with ~low ~tirring at 25C. ~extt 0~2 ml o beta-mercaptoethanol de~aturing agent is added and the solution i~ tirred slowly for 15 minutes at 25C. The solution is then allowed to stand under a nitrogen atmosphere for 16 hours at 25C in a stoppered fla~k. The closed environment is maintained by a pressurized nitroge~ tank constantly delivering ni~rogen into the flas~ with the ~lask vented in to a water trap.

30 15406`
~extr S ~1 of the solutiosl i s chromatographed on a Sephaaex brand G-15 ~el material colu~n which is a 12 inch x l inch column filled with gel. ~he column eluant i~ O o l M acetic acid at p~l 3 ~ The f low rate of the eluan~c through th~ columrl is ml/min. q~h~ ab~orb~noe of the outflow of the column i~
rnonitored at 276 nm by an ultraviolet detector. A gro~s protein ~raction of S ml is csllectedD q~he fra::~c;on is analyzed on a B~ckman Instrumen~ Company ACTA III ~;pectrophotometer., The s~olarity of the collected protein fraction i8 determined as 6.64 x 10-5 wh~rein the 280 absorbence i~ n .63, the molecular welght of 'che protein is 1300û daltons and the ~xtinction coefficient value, E, is equal to 7.3 for a l9~ solution.
To the S ~1 of protein ma~erial collected above is ~dded 0 . 5 ml of l.0 ~I tris-8Cl buffer, p~ 8; 0.5 ml of 1 mM E~T~; 0.5 ml of 5 ~c 19-4 ~ reduc~d gluta~hione; 0.5 ml o~ 5 ~ 10-4 ~
oxi~lzed glutalthione and 0.5 ~l o 0.02 M trypt~mine inhibitor for ~ter~e enzy~e. ~I!he 'c~eyplta~ine i~ added as an inh~bi or ~h~le the 2~D~A and gltltathione are added in ac:cordan~e wi'ch the teachirlg of Llr)ya C~avez, Jr. a~ld E~àrold Scheraga ~n ~Folding of Ribonuclea~e, 8-Protein ar3d De~ 51~1-124) ribonucleas~ ~uring Glutathione Oxldation of Reduced Prote~ns~ 80, lg, 9~6-1004 At ~h~ end of the 4O0 hour period7 resultant solution show~
~ pl~ of 5 Ø After ~t~ rring the reslaltant ~olution for 0 .
2~ hour~, at ~h~ pEI 5 value, the pFI is rais~d to 8 wi~h the ~ddition o~ 100 ~g of tris based cry~cals. The solu~cion is ~tl~red 81GI~1Y at p~ 18 and 25~ for 4 hour~O During th~
~tirrlnç~ proces~ a fluffy white precipi ate forn~s~
At the ~nd of 'ch~ 4 hour period, resulting solution i~
dialyzed ag~in~t l ~M tris gCl buffer, at p~ 7, conl:aining 2 .
tryp~amin~ inhibitor. A Spectropor brand membrane diaïysis .

9~

tubingl, No. 2 ~iz~, with a molecu}ar weight cu~off range of 12-14 ,000 dalton~ used for the dialysi30 The dialysis is conducted with two buffer ~hanges or a gro~ buffer volume of 7000 ml over a 17 hour period, a~c 0-5C~. wi~h slow shaking at 0 5~C.
Two ml of the protein ~olution above is chromatographed across a Sephadex brand G-15 gel column which is 1~2 inch x 12 inches using as an eluant 1 mM ris-~Cl buffer, at p~ 7, The eluant flow of eate is 1 ml/min. The absorbence of the eluting solu~ion i~ monitored at 254 nm. ~he first half of the eluting protein fraation is collected and assayed against esterase substratet namelyr tryptophan methyl ester ~TME) purchased from ~igma Chemical Company, lot lO9C-0û48 ~ l~o. T-5505 .
The assay for enzymatic activity of the newly syn~he~aze~
e8 erase enzyme-like modified protein prepared acc~ding to '~e present inventiorl i~ perforsned on a ~PI-C sy~tem. The column i~
packed with a upport material ~hich i a porous silica bonded pha~e material having carboxyl ~ide chains~ The column eluant is O . 03 M acetate at p~I ~, The column i8 of ~tainleæ~ steel composition and ~ mm x 25 em in size. A flow rate o 4 ml/min is main. ained ~y a conventional high pre~ure pump~ for ~ltample an Al~cex, Nodel 110A PUmPO The column eluarlt i8 de'cected by a conventional ul raviolet monitor a~c 254 nm and the tryptophan peak heiyhts are rec:oraed.
The as ay s~mple is prepared a~ follows. Three ml of the e~terase enzyme-like mcdlified protein (with a 280 absorbence of 0.45û at 280 nm, equaling 0,67 m~/ml) i~ admixed with 6 ml o 1 m~q tris~ l buffer, pH 7i.5 and 1 ~nl of 0~ T~E substrate. An assay control solution i~ prepared by adding 6 ml of 1 mM tris--- 2~ --~Cl buffer9 p~ ?.5, to 1 ml of T~E substrate and 3 ml of the Sepha~ex brand G 15 eluant buffer~ na~ely, the 1 m~ tris buffer,at p~ 7. ~he p~ of both the control and the as~ay sample is 6.4.
One ml of the ~ample is remoY~d from the beaker, usin~ a 2 ml. hypodermic syringe~ with an 18 ~auge needlea The one ml of ~ample is pa~æ~d through a 20 microliter injection sample loop onto the ~PLC column~ The injection time is recorded. This procedurP ig repeated using the ~ontrol solution. Both sample and control ~re chromato~raphed 4 ti~es in order o obtain a plo~ of ~olari~y of tryptophan verqus time for sample and control ~o elu~e from ~he ~olumnO
A ~tat~tical analysis i~ perfor~ed wi~h the data obtained from the abov~ procedure which shows a slope of 0~81333 x 10-5 M/min~ J with an error of 0.1021 x 10-5 is determined for the 8a~ple. A ~lope of 0.54062 x 10-5 M~in. with an error of 0.032gl ~ 10 5 ~/min. ~ deter~ined for the oontrolO
~ h~ foll~ing expre~sion is used to oalculate the ac~ivi~y of th~ es~ra~ enzyD~-like modified pro~ein according to the present inv~ntion.
A~t~v~y ~

~herein: change in slope is the differen~e between the s~mple and control ~lop~ (in thi~ example 0~272713);

e~ror i8 tb~ ~um o th~ errors in the sample a~d 5 co~trol ~lope~ (in thi~ example 9.1341; ~~s is the sampl~ volu~e in lit~rs; 106 is ~icromoles/mole; and ~p i~ ~g of ~odiXi~d protein in th~ assay.
In th~ above calculation, the concentxatlon of the modified .30 protein i~ calculated fro~ its ab~orbance a~ 280 nm, using the ~xtin~tion ooeffi~ient for ribonucleaset which is 7.3 or a 1%

9~r3~
154~6 solution. The extinction coeficient is disclo ed in the reeren~e ~nti~led 59 4~-62 (1973) author~d by D. ~. ~irschenbaum.
The assay result~ are as follows-Sub~trate T~ (Unlts~g) Initial activity 0~000 F~nal activity . 13~62 ~ 6,62 The re~ults sho~ that ~he e~tera~e enzyme~like modified pro~ein prepared according to the pr~sent ~nvention exhibits a~tivity wi~h re~pect to the e~tera~e enzyme su~trate ~ME where no activity is previously detected ia native ribonuclease enzyme, namely~ a nuclease to a second genus of protein, nam~ly, an estera~e enzyme-like modiXied proteiQ.
~X~PL~ 3 One hsndred my of boYine ~erum albumin (BSA), purcha ed from 5ig~a C~emi~al Company~ lot 90P-9315, ~o~ A-7511~ is dis~olved in 5 ml of 8 ~ urea containing Q.l M tris buffer p~ 8, ~ith ~10N
~tirring at 25~C~
Mext, 0.2 ~1 o beta-mercaptoethanol denaturing a~nt i addea to ~he solution. Th~ ~olution is shaken ~lowly for 15 minu es at 25C. The solution i~ next place~ under a nitrogen atmo~phere or 16 hours at 25C as di~closed ~n ~xample I~
- Next~ 5 ml o the ~olution is chromatographed acro~ a gel 25~ filtra~ion colu~n~ containin~ Sephadex brand G 15 gel filt~ation mat~rial. The column is 12 inches ~ 1 inch and is fil led with ~eph~dex brand ~-15 ~ Acetic aci~ at ~ol M, p~ 3, ~ used as the eluant for the gel ~olumn. The eluant flow rate is maintained at 1 ml/min via a ~low speed peris~alio pump of co~ventional design. An absorbence ~onitor is placed at the outfl :3w pOillt o~ the chromatography column and an u~ traviolet detector set at 276 nm is u~ed to detec~ eluting protein fractions.. .t~t ~he point whi~h protein starts eluting from ~he column collection is begun and a yross o~ 5 ml of ~?rotein is 5 collec~ed .
To the 5 ml of collected protein is added 1 ml of 1. 0 M tr is-~Cl bufer, p~ B; 1 ml of 1 mM EDT~; 1 ml of 5 x 10-4 ~q reduced glu athione; 1 ml of 5 ~: 10 4 Ml oxidized glutathione an~ 1 ml of 0 . 02 Pi tryptamine estera~e inhibitor . ~he resultant 10 solution shows a p~ of 5.2. The resulting solu$ion is s~irred ly for 0,,5 hour~ at pH 5n2~ After û.5 hours, tlle p~ of the solutior~ is raised to 6 wil:h 'che aropwise addition of 0.1 M
NaO8. The 801ution at pEl 6 is allowed 'co stir slowly at 25C
~or an additional 4 hours. I)uring the stirring process, a 1~ fluf~y white precipita~e ~3 formed. At the end of the 4 hour p~r~od the ~olution is dialyzed again~t 1 rDM l:ris-ElCl bu~er; p~I
7 t containing 29~ tryptamine inhibitor. The tryptophan ls added as the inhlbltor while other reagent~ are added as ~aught by Chavez et al di~cuss~d above~, The dialysi~ is per~ormed using a 2C dialy~is ~em~r~rle tubing, namely~ Spectrapor brand dialysis ~ubing~ ~o. 2 ~ r h~vlng a ~olecular ~eight exclusion range of 12~14,000 daltons. The dialysis procedure ~nclude~ 2 huffer change~ with a gro~ buf~er volume of 7000 ml of buff~r over a - 17 hour period, at 0050C, with low sha3cing of ~he dialysis 25 tubing in the dialysis media.
After dially~i~, 4 ml of the e~terase enzyme-like modii~ied protein containin~ 301ution i~ ~hromatographed across a Sephadex brand G-10 colu~n~ the columsl dimensioll being 1/2 inch x 12 inche~ and filled with gel agent usin~ tris-~Cl buffer at 30 p~ 7 as the eluant.. The eluant outflo~ing f!rom f10W rate i~ 1 ml~min. The @luant oi~ the column is monitor.ed at 254 nm to detect eluting enzyme-like modified protein.. A fir~t portion o~

eluting ~nzyme-like modified protein is collected and as~ayed again~t substrate for e~terase enzQme. The substrate used is tryptophan mel:hyl ester ~TM~ he ~ubstrate being. purcha ed fro~ Sigma Chemical Company, lot :L09C~0048, No. T-5505.
The activity of the estera~e enzym~-like modifled protein is te~ted as follows. The proteirl fra~tion colle~ted above is a~sayed b~ PLC to determine acti~ity toward the TME esteras~
~ubstrate. The assay conditions are a~ follows. Tlle ~PLe col~mn eluant i8 0.03 ~ acet~te~ p~ 6. The ~olumn ~upport material is a porous sili~a bonded phase material containing carboxyl side chains. The column is ~tainles~ ~teel composition and i5 2 mm x 25 cm~ with an eluant flow rate of ~ ml/min.
maintained by a conventional high pressure pump, or example an Altex, P~odel llûA pump. T~ column eluant is detect~d by a conventional ultraviol~t monitor a~ 254 nm and at 0.05 A.
The asæay ~ample is prepared as follows~ Five ml of 0 ~03 ~q acetate bufer, p~ 6, i~ ~nixed with 1 ml of 0,.1 ~ and with

4 ml of the e~terase enzyme like modified pro!ceill prepared above (with an adsorption at 280 nm o~ 0.016 equalirlg 0.û24 mgJml3, The assay ~ontrol is prepared by adding 5 ml of 0.03 M acetate p~ 6 t~ 1 ml of 0.1 M TM~ and to 4 ml of the eluant, namely" the ~ris-~Cl buffer, p}~7. Both the con~crol solution and the assay sample solution show p~ 5 . 8 .
One ml o~ th~ sample is removed from the bealcer, u~ing a 2 ml hypsdermic ~yring~, with an 18 gau~e needle. The one ml o ga~nple i~ pa~sedl ~hrough ~ 20 mlcrollter injeetion sample loop on~o th~ ~PLC column. The injection t~me is recorded. This procedure is r~peated using the control soiution. Both sample and control are t~hromatographed 4 imes in order ~o ob~ain a plot of molarity of tryptophan versus time for sar~ple and g~ 15406 control to elu~e f rom the column.
A ~t~tistical analysis is performed with the dat~ obtainea ~rc:m 'che above procedure whic:h shows a slope sf ul.055g6 x lQ-5 M/min., with an error oi~ 0.00213 x 10-5 M/min. is determil ed for the sample. A slope of 0.6244857 x 1û-5 M/min,. wilth an error of 0.000823 x lû-5 M/min. i~ determined for the control.
The following expression is u~ed to calculate the ~ctivity of th~ esterase en~yme like laodiied protein according ~o ~he pre en~ invention.

Acti~it~ = _ IgP
whereirl: Change in 510pe iS the difference between the sample and ~ontrol slopes ( in ~his example 0 . 0314);
error is the sum of the errors in ~he sample and control slopes ( in this example 0 . 00296); Vs is the ~ample volume in liters; 106 is micromoles/mole; and MP is mg of ~odified proteirl in the assay.
Xn the ~bove calculation, the concentration of the modified pro~cein is calculated from its ab~orbance at 280 nm~ using the extinction coe~fi~ient for ribonuclease, which is ~.62 f<3r a 1%
~olution. The extil ction coefficient is disclosed in the referenc:e enti'cled 3~3e~ 5, 49- 62 (1973) authored by D. M. Rirschenbaum.

The assay results are as follows:
Substrate TI~E ~nits/g ) Initial Activity 0.00 Final ActiviJcy 3~.7 3.08 ~he results show that the esterase enzyme-like modified pro~ein prepared ac~ording to ~he present inven~ion exhibits a~ivity with respect to ~he esterase enzyme substrate T~E.

~ o esterase activity is previously detected in the native BSA protein.
This illustrates the conversion of one genus oE nonenzymatic protein, an albumin, to another genus of protein, an enzymatically acti~e esterase enzyme-like modified protein~
,:~,q 3~

PXAMP~E 4 Two hundred fifty mg of BSA, purcha~d from Si~ma Chemical Company, lot 90F-8351, ~o~ 7511t is dissolved in 25 ml of 8 M
urea with slow stirring, at p~ 7.8 for 2 hours at 25C. After the 2 hour s~irring period, 1 ml of 10 mM beta--mercaptoethanol reduoing agent solution is added. ~h~ resultlng solution is ~tirred slow~y f~r 1 hour, at p~ 7~6, at 25C.
~ ext, 75 ml o a 1% ~olution of tryptamine e~terase enzyme inhibitor is added. The ~olution i~ slowly ~tirred for 2 hours, at p~ 7Ø
Next; the protein is cros~ linked a~ follows. The ~olution containing the protein is placed in a dialysis bag which is ~pectrapor bra~d dialysis tubing, ~o. ~ type. The prot~in contained in ~he dialysis bag i5 ~ialyzed against 1% tryptamine inhibitor solu~ion in 1 m~ ~ris buffer a~ pR 7.0, ~t 0-5C for 17 hour~. -~ he re~ultant estera~ enzyme~like ~odified pro~ein i~
purified as follow~i Two ml of the preparation abov~ is chromatographed acro~s a Sephadex G-10 chromato,raphy column usin~ 0.03 M ace~ate buffer p~ 6~ ~s the eluan~ ~he eluant flow rat~ i~ 2 ml~min. ~he initial half of he protein peak is approximately ~ ml of eluant volum~ and exhibits an absorbenre of 0.666 at 280 nm~
The a~ti~ity of the ester~se enzyme-like modi~ied protein prepared above is tested a~ follow~. The protein fra~tion collected above is assayed by ~PLC. The ~olumn ~upport material is porous silica bonded phase ~aterial containing carboxyl side chai~s. The column i~ stainless st~el composition and is 2 mm x 2S cm. The eluant flow rate is 4 ml/min. using 0~03 M acetate, p~ 6~

The column elua~t pro~ein fractio~ is detected by a conven'cional ultraviolet mon~tor at 254 nm.
The assay sample is prepared as follo~s~ Five ml of 0 . 03 M
acetate~ p~ 6~ i~ a~mixed with 1 ml of 0~.1 M T~qE and 4 ml of ~:he

5 proteln containing solution above. The assay control solution i~ prepared by admixing 9 ml of 0.03 P~ acetate, p~ 6, with 1 ml o~ 0.1 M T~qE substrate. The pH o:E both th~ control and assay sasnple solution is 6.û.
One ml of the s mple i~ remo~r~d from the beaker, using a 2 ml hypodermi~ ~yringe, with an 18 gauge needle. ~he 1 ml o sa~ple i~ injec~cea ~hrough a 20 microliter injection ample loop onto the ~PLC column- The inj8C~ l time is recordedO This pro~e~ure i~ repeated using control solution. Both sasnple and control are chro~atographed 4 times in order 'co obtain a plo~ of 15 mol~rity of tryptophan ver~u~ time for sample and control ~o ~lu~e f ~o~ the column .
A ~tatl~tical analysi~ is performed wi~h the data ~btaine~
~roFo the abc~ve procedure which shows a slope o 0.0516 x 10-5 M/~a$n~, with an ~rror of 0.004 ~q/min :Eor the sample~ ~ ~lope of 0.,0287 ~ 10-5 M/~in with an error of 0.0t)16 x 10-~5 M/min for lthe co~trol.
The follow~}lg e~pre~sion i8 used to calculate the activity of ~he e~t~ra~e en~y~ lilce modified protein according to th~?
pre~ent lnve~stion.
2~ v~ty ~ _ ., . ~P ~
~her~in: e~ng~ ~n ~lope i~ the difference ~etween the sample ~s~d control slope~;
~rror i~ the ~ùm o~ the error~ in the sample and the control slope;
Vs ~s the sample volume in liters;
10+6 ~S laicromole~/mole; and MP is mg of modified protein in the assay.
In the above calculationr the cvncentration of the modified .

protein is ~alculated ~rom its absorbence at 280 nm, using the extinction coefficier~t for bovine serum albumenr which is 6.6280 for a 1~ ~olution. The above procedure ~s disclosed in the reference entitled ~n~ J.rs~~rotein Res. 5 t 49-62 ~1973 ) _ _ authored by D. M,. Kir- chenbaum.
The assay results are as follow~.
Substrate TM~ ( Un i ts/g ) Initial Activity 0 v 0 10 Yinal Activity 0.57 ~ v16 The results show that the modified esterase enzsrme ~ like protein prepared according to the present invention e~l:hibits activity with respect to est~rase enzyme substrate TME. No a~tivity with respect to e~terase sub~trate TME i~ previ~u~ly detected in he native BSA.
This il~u~trates ~he ~onversion o~ one genus of .non enzymatic protein, an albumin tl~ ano~her ~enus of protein~
namely j an en2ymatically active ~s~er~ enzym~-like modii~ied pro~c~in ,, ~X~PLz 5 To demon-~tra-e that native bovine ~2ruDn albumin ~BSA~
protein shows no ~atalytic activity wi~h re~pect to the L-'cryptophan methyl ester (T~qE) ~ubstrate of Example 4 above~ the following, control procedure is perform~dv One g of BSA, from Sigma Chemical Compan~, Type `7511, lot 90~-9315, i~ di~;solved in 25 ~1 of distilled deionized water, with 810~ stirrin~ at 25C. To this solution is adde~ 75 ~1 of a 19~ tryptamine RCl solution. ~rhe re~ulting solution is incubated for 15 minutes at 25~C. This solution is next 30 dialyzed in a ~ilo~ 2 sized Spectra/PorR dialysi~ tube against 350û ml of a 1~ 'cryptamine-HCl solution for four hours. The -- 3~ --No. 2 tubing ha~ a molecular weight exclusion range of 12-14~000 daltons. The e~peratur~ of the ~olution is maintained at 0-5C. and the p~ at 6.0 during ~he dialy~is procedure. After the four hour dialysi~ procedure, two ml of the solution is chromatographed`a~ross a Sephade~ brand G-10 gel chromatography column. The ~olu~n is elu~ed with 0.03 M acetat~ buff~rt p~ 6, .
at a flow ra~e of 2 ml/min. The column size is ~5 inch x 1/2 inch.
~wo distinct peaks are collected. The firsS peak is eluted 10about 30 ml. ~he firs~ peak i~ identified by a 300-200 nm scan on an ACT~ 3 Spectropho~ome~er (Be~kman In~trumen~s Company~ as bein~ BS~ protein. The second p~ak i5 eluted at 60 ml ~nd is identified by ultraviole~ spec~rophotometry a~ tryptamine~
Four ml of the ~ollected BSA is ne~t assayed versus TM~ -~ub~trate for po sible enzymatic activity. The as~ay is conaucted a~ follows. ~ high pre~sure liquid chromatography ~y~tem i8 U~Qd for the a88ay. The eolumn is a 2 mm x 25 c~, ~tainl~s~ ~t~l colu~n packed with porous silica particle~
contalning carboxyl ~ize chain. ? with an av~rage partic}e pore ~ of ~0 microns, purchased ~rom Baker Ch~mical CompanyO
The colu~n ~luant i~ 0.03 M acetat~, p~ 6, at a flow rate o 4 ml/~ln. ~he absorbance of tbe outflow of the column is ~onl~or~d at 254 ~m. Five ml of the 0.03 ~ a~etate, p~ 6 and 1 ~1 o~ 0.1 ~ TME $ubstrate ar~ mixed with 4 ml of the BSA
solu~ion above.
The a~ay control $g prepar~d by mixing 9 ~1 of 0O03 M
~e~ate~ p~ C ~nd 1 ml of 0~ M2 ~ubs~ra~. The p~ o bo~h the control and 5a~ 6Ø
One ~1 o~ the a~say s~mple is removed from a sample beaker with a 2 ml hypodermic ~yring~ with an 18 gauge needle and 15~0~
injected through a 20 microliteY injection sample loop onto the column. The time o~ injec~cion iæ re~orded.
The procedure i~ then repeated using the a~say control solutis~n. Both the ~ample solutiorl and control solu'cion are chromatographea four times in s:~rder to obtain a plot of molarity of TME versus time foe the assay sample and the assay control ~;olution~ .
A statistical analysls of the data obtained ~how~ a ~lope o 0.19689 x 10 5 M/mins wi'ch an eEror oiE 000089 x 10-5 M~min for the 13SA assay sample. For the as~ay control ~ample, a slope of 0.20296 x 10-5 M/min, with an error of 0.0062 x 10-5 M,~min is determined. The slopes, plu8 the sums o~ the errors, are virtually identic~l. Accordingly, the natiYe BSA prote~n ~hows ns:~ measurable catalytic ac~ivity prop2rties with reslE?ect to TME 8ubstrates.
~XAMPLB 6 Ts~ demonstrate that native ribonuclease enzyme show~ no catalytic activity with r~spect to L-tryptophan ~thy~ ester (TMS) su~skrate of Ex~mples 1~2 above, the following eontrol is performed .
Sixty mg of ribonuclease enzyme, from Sigma C:hemical Company~, Type R-5000, lot 20F-2010, is dis~ol~ed in 100 ml of rnM tri~ buffer, at p~l 7 9 with slo~ ~tirring a'c 25~C.
~rhe rii:~onuclease c4ntaining solution is then dialyzed using a ~c~. 3 S3?ectra/PorR dialysis tu~e agains~ 3500 ml oiE 1 ~M
tris buffer, p~ 7, for 17 ~ours at ~ 5~. The NC~r 3 tubing has a molecular weight exclusion range of 35-4500 ~altons. ~ext, 1 ml of the r~bonuc:lease con'caininy solution is assayed versus TME
subs~rate for ps:~ssible ~nzymatic activity with respect 'co TMB.
The assay is corlducted as follows.
A high pressure liquid chromatoyraphy system is used for the assay. The column is a 2 mm x 25 cm stainless steel column packed with porous silica containing carboxyl side chains having -- 3~ ----an average pOrQ ~ize of 40 microns, purchased from Baker Chemical C:ompany. The column eluan~ is 0O03 M acetate, pH 6, at a flow rate s~ 7 Isll/minO The ab~;orbance of the column outf low i~ monit~rea at 280 nm~
The assay Eample solu'cion containing native ribonuclease is prepared as follows. Eight ml of one m~ tris buEfer, p~ 8.05, ~ admixe~ with one ml of Qol M ~rME, pR 3"4 and 1 ml of the r ibonuclease æolution prepared above. The assay control ~olution is prepared by admi~Ring 8 nil of 1 mM tris bu~fer, pH
8~05; one ml of 0.1 M T~E:, pEI 3.4 and one ml o one mM tris buffer, p}~ 7Ø The p~ of ~he conJcrol and assay ~olutions is S . 5 O
~e~ct; one ml of the assay sample solution ls removed from the ~ample beaker using a 2 ml hypodermic syringe with an 18 gauge needle and injected onto the column through a ~0 microliter in~ect~on sar~ple loop. The time of injection ~,5 recorded .
Thi~ pro~:edur~ i~ then repeat~d using the ass~y t:on'crol ~olution., 13Oth ~a~ple and control ~olutions are chromatographed four ti~e~ :Ln order to obtain a plot of molaril:y of TMI~ ver~us tl~e for ~a~ple ~nd s:on'erc)l ~olutions.
A statistieal ~nalysis of 'che data ~btained show~ virtually identi~al 11 ope~ of 0 .1400 t O .003 ~or both corl~rol and sample solu~cions. Accordingly, t23e na~ive rlbonucleas~ ~hows no 25 loea~urable catalyll:ic ~c~ivi~y properties with r~sE:ect to the T~E
~ub . ~rate.
EX~IPLE~ 7 To demonstrate that natiYe bovine serum albumin (BSA) a~:tivity w~th r~spect to L-tryptophan methyl ester (TME) 30 subs~rate of E:x~mple 3 above, the following control is .

per f ormed O
One hundred mg of BSA ro~ Sigma Chemical Company, No. 7511, lot 90F-8351, is dissolved in 100 ml of one mM tris bu~fer at pH
7, with ~low stirring at 25C.
The BSA containing solution is then dialyzed usin~ a No. 2 Spectra/PorR di~lysis tuhe again-~t: 3500 ml of 1 mM tr is buffer, p~ 7, ~or 17 hours at 0-5"C~, ~he ~o. 2 tubing has a molecular weight exclusion range of 12-14,00û dalton~ ext, one ml of the BSA containing ~olution is as~ayed versu~
substrate ~or possible en~ymatic activity with respect to TME.
The assay i~ conduct:ed as follows.
P~ high pressure liS~uid chromatography system is u~ed ~or the assay. The oQlumn is a 2 mm x 25 cm stainless ~teel column packed wl~h porous ~ilica containing ~arboxyl side chaln~ hav~ng an average pore ~ize of 40 micron~, purchased from B~k~r Chemicai Company. The column eluant is 0.03 M acetate~ 6, a~
a ~low rate of t ~l/min~ The absorbance of the coluDI;~ outflow is moni~ored at 280 rlm.
rrhe assay sample solution containing native 13SA is prepared as follows. Eight ~1 of on~ tris buf~er~ pE~ ?.5 1~ admixed wi'ch one ml of 0.1 ~ TME; p~ 3.2 and one ml oiE the ribonuclease solution prepared above. The assay control ~olution is prepared by admi~ing 8 ml of one raM tri~ bu~er~ p~ 7.5, one ml of 0~1 M
T~, pEI 3.2 and one ml o~ one mM tris buffer, p~ 7.û. The p~ of the control and assay ~olu~ions is 5.8.
~ex~, one ~al oiE the as~ay sample ~olu~ion ~ rernoved from . the sample beaker using a 2 ml hypo~ermic syringa with an 18 gauge needle and injected onto the column through a 20 microliter injection sample loop,. The time of injection is recorded.
This procedure is then repeated using the assay control .~ Q:~9~
15~06 801ut:~0n,. Both sample and control solutions are chromatographed four times in order to obtain a plo~ o:E molari~cy of TME versus time for ~ample and cc>ntrol solutions.
A s~ati~tical analysis o~ the data obtained shows virtually identical slopes of 0.06 ~ ~0065 for bo h control and sample ~olutions. Acc~rdingly, 'che native ribonuclease shows no measurable ca~alytic activity properties with respect to the TME
~ub~rate.

Claims (28)

WHAT IS CLAIMED IS:

1. A process for chemically altering the substrate specificity of a native protein to produce an enzyme-like modified protein, comprising:
a. selecting an enzyme to be modeled;
b. grossly denaturing a native protein;
c. partially renaturing said grossly denatured native protein to produce a partially denatured protein;
d. contacting said partially denatured protein with an inhibitor for said model enzyme to form a partially denatured protein-model enzyme inhibitor complex; and e. cross-linking said partially denaturad protein in said protein-inhibitor complex.

2. The process of claim 1 wherein said native protein is grossly denatured by forming an aqueous solution of said native protein and maintaining said aqueous solution at a temperature and for a time sufficient to grossly denature said native protein.

3. The process of claim 2 wherein said temperature is between 50 and 70 degrees centigrade.

4. The process of claim 1 wherein said native protein is grossly denatured by admixing said native protein with water to form an aqueous solution and admixing the resulting aqueous solution with a denaturing agent.

5. The process of claim 4 wherein said denaturing agent is an organic acid.

6. The process of claim 5 wherein said organic acid is selected from the group consisting of acetic acid, formic acid, propionic acid and citric acid.

7. The process of claim 4 wherein said denaturing agent is an inorganic acid.

8. The process of claim 7 wherein said inorganic acid is selected from the group consisting of nitric acid, phosphoric acid, sulfuric acid and hydrochloric acid.

9. The process of claim 4 wherein said denaturing agent is guanidine hydrochloride.

10. The process of claim 4 wherein said denaturing agent is urea.

11. The process of claim 4 wherein said denaturing agent is a mixture of urea and beta-mercaptoethanol.

12. The process of claim 1 wherein said partially denatured protein is cross-linked by contacting said protein with a cross-linking agent.

13. The process of claim 12 wherein said cross-linking agent is glutaraldehyde.

14. A process for chemically altering the substrate specificity of a native protein to produce an enzyme-like modified protein, comprising:
a. selecting an enzyme to be modeled;
b. grossly denaturing a native protein;
c. admixing said grossly denatured protein with an inhibitor of said model enzyme;
d. partially renaturing said grossly denatured protein, in the presence of said inhibitor, to produce a partially denatured protein inhibitor-complex; and e. cross-linking said partially denatured protein portion of said protein-inhibitor complex.

15. The process of claim 14 wherein said native protein is grossly denatured by forming an aqueous solution of said native protein and maintaining said aqueous solution at a temperature and for a time sufficient to grossly denature said native protein.

16. The process of claim 15 wherein said temperature is between 50 and 70°C.

17. The process of claim 14 wherein said native protein is grossly denatured by admixing said native protein with water to form an aqueous solution and admixing the resulting aqueous solution with a denaturing agent.

18. The process of claim 17 wherein said denaturing agent is an organic acid.

19. The process of claim 18 wherein said organic acid is selected from the group consisting of acetic acid, formic acid, propionic acid and citric acid.

20. The process of claim 17 wherein said denaturing agent is an inorganic acid.

21. The process of claim 20 wherein said inorganic acid is selected from the group consisting of nitric acid, phosphoric acid, sulfuric acid and hydrochloric acid.

22. The process of claim 17 wherein said denaturing agent is guanidine hydrochloride.

23. The process of claim 17 wherein said denaturing agent is urea.

24. The process of claim 17 wherein said denaturing agent is a mixture of urea and bata-mercaptoethanol.

25. The process of claim 14 wherein said partially denatured protein is cross-linked by contacting said protein with a cross-linking agent.

26. The process of claim 25 wherein said cross-linking agent is glutaraldehyde.

27. The enzyme-like modified protein product of the process of claim 1.

28. The enzyme-like modified protein product of the process of claim 14.