Purification of enzymes by heparin-sepharose affinity chromatography

PmCAmON

OF

ENZYMES

BY

KEPARZN-SEPHAROSE

AFJ?IMTY

CHROMATOGRAPHY AKELAQ

A. FAROOQUI

Depmtrrentof BiixkenuMys Uiriversityof Georgia. Athens. GA 30602(U.S.A.) (Received March 3rd. 19SO)

1. wrodu~on . . . . _ . _ . _ . _ . . . . _ . _ . . . . . . _ _ . 2. Interaction of hep&n with enrymes........................ 3. Methadsforffiecovalentattachmentof~~toSep~ose. _ _ _ _ . 4. Pllrzcation of enzymes by hepar&Sephmxechmmatogsaphy _ _ _ _ . S.Otherusesofhe~p~~chro~t~phy.. . . . _ . . _ . . 6. Advantages and disadvantages of heparin-sephamie chromatography . _ 7.Gmchlsion ._____.._._......._....-*.-...... 8_SrJmmary_._..._.._.......................... References __._-_.__.___._._._..-.-.* _.........

_ . _ . _ . _ 335 337 _ _ _ _ _ _ . 338 _ _ _ _ _ _ _ 340 _ _ _ . _ . _ 341 _ _ _ _ _ _ _ 342 *. 343 343 343

1. INTRODUCTiON

In recent years the plurificationof enzymes has been revolutionizedby the This method utilizedthe specificinteractions techniqueof afhnitychromato,%phyLm2. found in biological systems.Most proteinsundergobiological interactionswith other proteins and small molecules in order to function. Thus, enzymes bind with substrates, inhibitors, coenzymes and allostericelectors; antibodiesinteractwith antigens; hormones bind with specik binding proteins and ceklat receptors; lectins bind carbohydrateand nucleic acids and repressorproteins interact with genes3s4. These interactionscan be used for the puriGc&ionof proteins. The general scheme of &in&y chromatographyinvolves the covalent attachment of a l&and to a solid support. A crude tissue or bacterialhomogenate is then applied to the columu; only the protein that spcciticallyinteractswith the ligand will be retained, whilst the other proteins, having no aEnity for the immobilized &and, will be washed out. The retainedprotein can be eluted specitkallyfrom the column either by adding an excess of ligand to the equilibratingbuffer or by using substancesthat interferewith the protein-ligandbindingsuch as salts or denaturants. The successof afhnitychromatographydependslargelyou how closely the conditions used in the experimentmimic the nativeor biological interactions.Careful consideration must be given to the natureof the matrixand to the stericrestrictionimposedby immobilizationof the l&and. The matrixshould ‘berelativelyhydrophilicand should possess good chemical and mechanicalstabilities.The stericeffect can be overcome by using spacers. Both matrix and spacer should lack non-.specSc absorption sites. The other importantpoint is the selectionof hgand. There are two classesof l&and% general hgands and specik ligands. General ligands interact with marrydifferent

Effect

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition

Enzyme

Acidphosphntasc Alkalinophosphntaso N=Acetyl.B.hoxosaminidase Acidlip&so CnthcpsinBandC a. and P-Fucosidaso u=and fl-Oolnctosidnao Hyaluronldnso a.Monnosidaso Ncuraminidnw DNApolymcnsc RNApolymoraso

TAIILE!1 DFFECI’OF HEPARINON VARIOUSONZYMES

::

1: 11 46

i: I1

11

12,45 12

Rel:

Ribonuclcanc Deoxyribonuclcaso Endonuclcasc Trypsin Pepsin Fumcraso Lipoprotoinlipnso Trchaloscphosphntcsynthctase Tyrosinohydroxylrrso

Etlzyttle

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Activation Activation Activation

16,17 47 48 20 21 22 24,26,27 32-34 35-37

Reji

PURIFICATION

OF ENZYMES

ON HEPARENSEPHAROSE

337

proteinsand examplesare NAD* Cibacron blue and 1ectin.s~‘. I&an& that interact with one or few proteins are called specificligands, examplesbeing antigenSg.Thus a&&y chromatographyis a uniqueseparationtool whichhas a major advantageover other methods of purification due to its tremendous specificity. This technique allows rapid purifkation of various proteins with good yield in a single step. 2. ENTERACTZON

OF EEJ?ARIN

WTZH ENZYSES

Heparin is a linear and highly sulfatedglycosaminoglycanwith anticoagulant properks. It exists in a wide range of polymers (mol. wt. 5000-3~,ooO) and is composed of disaccharideunits of a-r..-idopyran-uranicacid 2-sulfate and 2-deoxy-2sulkmino-a-D-glucopyranose6-suWatelinkedthroughpositions 1 and 4qs10.Due to its polyanionic nature heparin interacts with many enzymes (Fable 1) and produces signCant changes in their activities.Thus, it inhibits almost all lysosomal hydrolases”. On the basis of variation of ionic strengthand pH value, it was suggested that reversible electrostatic interactions between anionic groups of heparin and cationic groups of the enzyme moleculewereresponsiblefor this inhibition.Although the nature of the inhibitionof all these enzymes was not determined,the inhibition of acid phosphatase12and hyaluronidase13 was competitive.Heparin causes marked inhibition of enzymes associated with nucleic acid metabolism. Thus, it inhibits DNA polymerase14,RNA polymerase15and ribonuclease 16*L7. It must be emphasized here that the selective binding of these enzymes to heparin is not due to ionic interactionsbut rather due to enzyme-inhibitor imkraction. In the case of DNAdependentRNA polymerase, the heparin interfereswith the initiation of transcriptii~n’~*~.Other enzymes such as trypsin, pepsin and fumarase are also inhibitedby heparin, pro-bablydue to the formation of a heparin-enzyme complexzGz_ Here it must be recalledthat trypsin= contains an essentialimidazolegroup in its active site. It is possible that heparin inhibits trypsin by blocking the positively charged imidazole group which is essentialfor its activity. Heparin stimulatesseveralenzymes. K0rn2’*~ isolatedlipoproteinlipase from rat heartand showedthat the hydrolysisof chylomicronby this enzymewas activated by heparinand strongly inhibitedby a polycation such as protamine. He postulated that lipoprotein lipase contained bound heparin which could be competitively displacedby protamine. Patten and HoIlenbergz6suggestedtha%heparinstimulation of lipoprotein lipase was due to increasedbinding of the enzyme to chylomicrons and heparin had no effect on the stabilityof the enzyme OF on its activity once the complex was formed. On the basis of various kinetic studies, Whayne and Feltsz’ suggested that heparin may function as a specificligand that acts as an allosteric modifierof lipoproteinlipase and alters the kineticsof substrate-enzymeinteraction. Iverius et rJ.28also concluded that lipoproteinlipase containedsmall amounts of an endogenous, heparin-likematerial, father indicatingthat hepzrin may be part of, or bound to, the enzyme protein. Although the exact rBle of heparin in lipoprotein lipase reaction is not known, the collective &den-’ indicatesthat intravenous injections of heparin in the rat, pig and man cause the release of two lipoprotein lipases which diEer in their sensitivityto NaCl, pro&mine sulfate and diethyl-pnitrophenylphosphate and in their requirementfor a lipoproteinpolypeptide. One of these lipases is reported to originate solely in extrahepatictissues and requires

A. A. FAROOQUI

338

apoLp-Glu for maximal activity. The second lipase, res%tant to high NaCl and protamine sulphate, has been reported to come from the liver. Ir: titro, heparin stimulateslipoprotein lipase but the precise nature of this activation is not clear. Heparin not only stabilizesthe enzyme and protects it from heat inactivation,but 21~0plays a r6le in linking the enzyme to its lipid substrate. Lap;, et aL3= were the first to report the sttisnulationof trehalosephosphate synthetaseby heparin-They found that heparinconcentrationsofO.5-I cle/mI were optimal for activation, whereas higher concentrations (5 pg/ml) were inhiiitory. Elbein and Mitchella*u reinvestigatedthe stimtiatory effect of heparin on the trehalose phosphate synthetaseactivity of Mycuhcferti smegmctisand reported that in the presence of heparin (OS mg/ml) the trehzdosephosphate synthetase shows increasedstability when heated at WC for various time periods as compared to the enzyme in the absence of heparin. Heparin also prevents digestion of the enzyme by trypsin. In the absence of hepariu, the enzyme was retained on a SephadexG-200 cohunu (ruol.wt.40,000-5QOOO).However,whenheparin(0.5 mg/ml) was mixed with the enzyme the trehalose phosphate synthetase was excluded from the SephadexG-200 column and eluted in an area suggestinga mokcular weight of greater than 450,000. The data of EIbein and Mitchellu were consistent with the vie= that hepariu stabihzesthe trehalosephosphate synthetaseand also causes an asso&ation of the enzyme to high-molecular-weightforms which have increased stability. The enzyme tyrosine hydroxylase is also activated allostericallyby heparin. Kuczenski and MandeU3s*s reported that the addition of heparin induces a conformational change in tyrosine hydroxylase which results in a greater aEnity for norepinephrine.It has also been suggestedthat heparincan produce a more active tyrosine hydroxykse which is responsiblefor modulation in the level of the end product, norepinephrine.Now it is believed that the normally soluble tyrosine hydroxykse becomes activatedas it approachesthe surface of the synaptic vesicle or other membranesite which contains heparin3’.The action of heparin or heparin sulfate on tyrosinehydroxylaseis not shared by the offier mucopolysaccharides. 3. METHODS

FO.7 THE COVALEbii

ATTACJiHMEN’F OF HEPARIN

TO SEPHAJXOSE

Two procedureshave been used for couphng heparin to Sepharose: (1) the cyanogen bromide procedure; (2) the cyanuricchlorideprocedure. IveriuP was the first to coupIe heparin to Sepharose 4B using cyanogen bromid&g**o_Sepharose 4B (settled bed volume 500 ml) was washed twice with distilled water by decautationand suspendedin 1.5 I of distilledwater. Cyanogen bromide (25 g) weredissolvedin 50 ml of cold dime*&ylformamide and added to the Sepharose s-uspensionwith stirring in an ice-bath. The pH of the mixture was maintainedbetween 10.5 and 11.5 with 5 N NaOH and the reaction was allowed to continue until the rate of change of the pH with time became negligible (about l-l.5 h). The suspensionwas then poured over a f&ted glass tiherand the activated Sepharose4B washedwith 8 1 of water AU of these operationswerecarriedout in a fme tz;pboard. The Sepharose4B cake was suspendedin I .5 I of 0.1 M NaHC03 containing 1.5 g of heparinand the suspensionwas stirredslowly overnightat 4°C. Triethylamine(60 n@ was then added and stirringwas continuedfor a fkrther4 h.

339

,

Fig. 1. Reaction sequence showing the coupling of beparin to Sepharose by cyanogen bromide procedUre.

The heparin-!%pharose was collected on a fritted glass filter and washed with 3 1 of water. The gel was then suspended in an equal volume of buffer containing 20 mM Tris-HCl buffer, pH 7.0, and 0.5 mM EDTA. The chemistry of the cyanogen bromide reaction is not known but it has been suggested~r that isourea or carbamate derivatives are formed during the reaction (Fig. 1). This procedure depends on the availability of free amino groups on the heparin molecule. The source of such groups is either an unsulfated hexosamine unit or a N-terminal amino acid retained from the native proteoglycarP_ The preparation of heparinized aminoethyl agarose using cyanogen bromide has also been reported”. Srivastava and FarooquP covalently attached heparin to Sepharosc 4B using cyanuric chloride (2,4,~trichloro-l,3.5-triazinz). Sepharose 4B (50 ml) was washed five times with 1 M Na,CO, and the excess of Na,C03 was removed by draining. Heparin (100 mg) dissolved in 5 ml of 1 A4 Na,CO, was added to Sepharose 4B with constant shaking. Cyanuric chloride (1 g) suspended in 15 ml of acetonitrile was added to the above Sepharose 4B. The mixture was maintained at bH I I with 1 M Na2C03 during shaking for 1 h at 65°C. The reaction mixture turned yellow during the reaction. After 1 h the mixture was filtered through a biichuer funnel, and washed with 500 ml of water-ethanol-trimethylamine (2:l: 1, v/v/v)_ Finally, the heparin-Sepharose 4B was washed with 50 mM Tris-HCl buffer, pH 7.0, containing 0.5 mM EDTA. The proposed mechanism of coupling by cyanuric chloride is shown in Fig. 2. During the activation reaction the free hydroxy group of heparin reacts with cyanuric chloride to give a heparin+zyanuric chloride complex at alkaline PH. The hydroxy group of Sepharose 4B then reacts with a second chlorine atom to form heparin-monochlorotriazinyl Sepharose 4B. The amount of heparin covalentiy bound to Sepharose can be determined by measuring the hexosamine and sulfate contents of the gel. Thus the cyanogen bromide procedure resulted in coupling of about Z mg heparin per ml of wet Sepharose 4B%. The cyamuic chloride procedure gave a heparin-Sepharose preparation which had 0.8 mg heparin per ml of the wet geP. Recently Varshavskaya et ~1.~ described a simple method for the determina tion of the heparin content in heparin-Sepharose. This method is based on the potentiometric titration of heparin with 0.5 M KOH. The

A. A. FAROQQUX

WEP-

Q”-“\\

o---c\ N=<

Fig. 2. The ixckxmisxnof coupling of kparin to Scpharose by cyamric ch!ori& proc&ureU.

values obtained for the coupled heparin by the potentiometic method and by the determinationof hexosamineand sulfate content were similar. I

PURIFICATION

OF ENZYMES

BY KEPARING3EPKAROSE

CMROKATOGRAPKY

The list of enzymes purikd by heparin-Sepharosechromatographyis shown in Table 2. The heparinGepha.rosewas washed with the desiredbuffer and packed into a small cohxmn. The crude enzyme preparation,dialysed against t&e column TABLE 2 ENZYMEtS PURIFIED

BY KEPARIN-SEPEFAROSE

AFFINITY

Pfm~cafion achieved (FoW Lipprot& L;psc DNA _ml~ =JAp&=== R&r&ion ~donuckse ~Jhz=== Iduronidasc KeparinN-sutp~ Kyalmok&KC Thnzhse~osphatesynthetase

107-12s lu-sO 10 7 18 4 4-10 la-38

Ref. 4%53 14 %!x 48 59 60 61 43.62 34

CKROMATOGRAPKY

equilibratingbuffer, was applied to the heparin4epharose 43 column at a flow-rate of LO-L2ml/h. The enzymeshaving affinityor interactionwith heparinwere retained and the rest of the proteinswerewashedout. The enzymes appearedas a sharp pe& when the column was eluted with a gradientof N&l or heparinor some other compound which interfereswith the binding of heparinsephahose with the retained enzymes, The heparinsepharose can be+ regenerated for rechromatography by washing the gel alternatelywith fifteento twene column bed volumes of buffersof : pH 8.0 and pH 5.0 containingOS M NaCl. It is obvious from Table 1 that heparin-fkpharose can be used for the purificationof lysosomal hydrolases, and enzymes of nucleic acid and lipid metabolism. According to Brennesselet al.“, heparinsepharose may be usefulin evaluating the subunit structureand mechanism of action of euharyotic DNA polymerases, particularlysince the inhibitionof at least one cellular DNA polymeraseenzyme by heparincan be correlatedwith a dissociationof the polymericform of the enzyme into smalleractive species63.The difkences in the molecularweightsof DNA polymerase isolated by heparinSepharose chromatographyand by conventional methods may help in determiningthe structureand function of these important cellularenzymes. Lipases having similarphysicochemicalpropertiescan also be separatedby heparinSepharosechromatography. Thus, Boberg et &.51 separatedlipoproteinlipase and hepatictriglyceridelipaseby this procedure.HepariQ-Sepharosechromatographyhas been successfully used for the separation of two forms of hyaluronidase43and heparin N-sulphata@. It must be noted here that it was not possibleto separatethese forms eitherby ionexchange chromatography or polyacrylamide gel electrophoresis.During the purikation of hyaluronidase C3*6*by heparin-Sepharose chromatography it was clearly shown that the binding of this enzyme to heparin-Sepharosewas due to enzyme-inhibitor interaction. As heparin-Sepharose is a polyanionic gel, it may interactwith many proteinssuch as lysotyme64and acrosin65. These proteins have isoelectric points of 10.5 and 10.2 respectively. It is interestingthat both these proteins are retained on the heparin-Sepharosecolumn and are readilyeluteclwith a linear gradientof O-1 M NaCl but not with heparinor hyaluronic acid. This strongly suggests that, unlike hyaluronidase,the binding of lysoqme and acrosin to the heparin-Sepharosewas due to charge interactions. 5. QTEiER

USES OF HEPARINSEPHAROSE

CHROMATOGECAPHY

Heparinbinds selectivelyand fkmly to many blood proteinssuch as fibrinogen, thrombin and plasmintie It also interacts with low and high density serum lipoprotcins67*6g. HeparinSepharose was successfullyused for the purificationof thrombina, autithrombinIIP”, arginine-richapoprotein71and low and high density lipoproteinsi2*tl. On the basis of acetylation of low density lipoproteins, Iverius72 suggestedthat lysineresiduesof theseproteinswereinvolvedin bindingwith heparin. In the case of arginine-richapoprotein”, arginine residueshave been shown to be important during ffie interactionof heparinwith protein. The fact that the heparinprotein complexes can be disruptedby high salt concentrationindicates the ionic nature of these interactions. Heparin also interactswith cytosol oestrogenreceptorof lactatingmammary

A. k

342

FAROOQuI

glands and utenrs7’. The treatment ofcytosol with heparin dissociates the oestrogenbinding activity from the 2ggregate into a receptor. Auricchio et QL’~ puri&d this receptor by heparin-Sepharose chromatography and suw that the heparininduced dissociation of the receptor could be due either to the removal of substances associated with the receptor or to the direct interaction of the receptor with heparin. Heparin-Sepharose chromatography was eEciently used for the purification of

androgen receptors from rat prostate76. It must be noted here that the nature

of the

interaction between these receptor proteins and heparin was not determined. It is hoped that some explanation of receptor-heparin interaction will be fo.rthcoming. The binding of heparin to other proteins also includes insulin, oxytocin, corticctropin and vasopressin”. Aborg and I_J~nas’~reported a protamin*hcparin complex which has the ability to bind histamine and monoamines by ionic interactions_ Several studies7es2 have indicated that heparin-Sepharose chromatography was efficiently used for the isolation of protein synthesis factors and ribosomes. The pollyribosomes retained on heparin-!Tepharose are readily dissociated into subunits by a mere increase in the Kf/Mg2+ ratio. The use of heparin4epharose chromatography for the separation of various protein synthesis factors will result in further characterization of initiation, elongation and protein synthesis factors and will result in considerable knowledge regarding the protein synthesis. It has recently been reported 83*64 that heparin-Sepharose can be used for the removal of kholesterol from the blood and can be important for the treatment of homozygous familial hyperchofesterolemia. 6_ ADVANTAGES GRAPHY

AND

DISADVANTAGES

OF HEPARIN-SEPHAROSE

CHROMATO-

Heparin-Sepharose chromatography offers many advantages over ionexchange chromatography. Heparin has a stable structure and once coupled the heparinSepharose is stable for at least 1 year. This gel has excellent flow properties and packs well into the column. The chemical stability of heparin-Sepharose appears to be quite good. Due to the polymeric and polyanionic nature of heparin, the gel has a very high capacity allowing the use of small columns. As 2 result the enzymes elute from the column in small volumes with very high activity. The proteins retained on heparin-Sepharose are very sensitive to variation of pH and ionic strength. Furthermore, the peaks of enzymes eluted by heparin are sharper than those eluted by a salt gradient. Heparin is a general l&and which interacts with many enzyme proteins. Some of these interactions are specific and others are ionic. The major disadvantage of heparin-Sepharose chromatography is its broad specificity. Thus it is not possible to obtain homogeneous enzyme preparations in a single step. If homogeneous preparations of enzymes are required, the heparinSepharose chromatography must be combined with other methods of purifications. The heparin_Sepharose cannot be used below pH 3.0 and above pK 9.0 because many proteins which bind to heparinSepharose between pH 5 to 7 are either not retained in the column or are irreversibly bound to the heparinsepharose matrix.

PURElCATlON

OF ENZYMES

ON HEPARIN-SEPHbJX?SE

343

7; CONCLUSION

From the above discussionit is clearthat heparininteractswith many enzymes and proteins. These interactionsmay result in activation or inhibition of enzyme activity. The fact that heparin CIIIbind to or alter enzyme a&ivities is perhapsnot surprisingsinceit is highlychargedat physiologicalpH values. The crucialquestions, whetherthese interactionsoccur in v&o and play some r6le in regulationof enzyme activities, cannot be answered at this time because our knowledge of heparinprotein interactions is still incomplete. However, there is a reasonable body of evidence to indicate that heparin does interact with cells or protein in connective tissue= and circuIatingsy~tem~.~. The “discovery” of heparinsepharose has been one of the most important developmentsin analytical and preparativetechniques used for the separation and isolation of various proteins. For the last 3-4 years, heparinhas been highly exploitedas a ligand for aEn& chromatography.Yet, only a small percentage of the total number of enzymes which interact with heptin have been chromatographedon heparin-Sepharose_It is hoped that in the futureheparinSepharosechromatographywiJ.lbe widely used for protein fractionation. 8. SUMMARY

Heparin sekctively interactswith many enzymes and brings about signiE=t changesin their activities.Due to its polyanionicnature, heparinalso binds to many cationic proteins. Both these interactionsmay be used as a basis for the purification of proteinsby heparin-Sepharoseafkity chromatography.Recently heparin-Sepharose bi3sbeen extensivelyused for the purikation of enzymes, coagulation proteins, steroid receptorsand protein synthesisfactors. REFERENCES P.Cuatrecas;as and C. B. Anfiasir, Amu. Rev. &&em., 40 (1971) 259-278. C. R. Low and P. D. G. Dean, in A#iizzXyChramatogmphy, Whey, London, 1974. C. R. Low, Int. J. Bochem., 8 (1977) 177-181. C. R. Low, in P. V. Sundaram and F. E&stein (Editors), 27zeoryUJZZ Praciice in AfFiry Tedniques, Academic Press, New York, 1978,pp. 55-75. 5 K. Mostrach, in 0. Hofhmn.Osterhof, M. Breite&ach, F. Keller, D. EZrai and 0. Weiner (Editors), Amm Chronz~ogrqhy, P-on, New York, London, 1978, pp. S-66. 6 L.A.KatfandR.L. Easterdzy, in P. V. Sundaramand F. E&stein, Editors) 27reory andpracrice in Afirn%y2iechn@es, Academic FESS, New York, 1978,pp. 23-44. 7 J. T. Dukmey, Mol. Cell, Biochem., 21 (1979) 43-63. 8 T. ES-isin 0. Hoffmann-osterhof, M. Btitenbxh, F. Keller. D. Kr& and 0. !Scheiner (Editors), A&ry Cknxnafography,Pergznon, New York, Oxford, 1978,pp. 191-210. 9 J. Kiss, in V. V. l&kIcar and D. P. Thomas (Editors), Heparti Ciiematry and Clinical Usage, Academic Prss, New York, 1976,pp. 3-10. 10 A. Linker and P. Hovingb, in N. M. McD&ie (Editor), Hepark: Srructure Ce&hr FwrL.tion mrd Chizica!AppZcat&vz.ss Academic Press, New York, 1979, pp. 3-24. 11 J. L. Avilaad J. Cbkt, B&x&m. J., 160 (1976) 129-136. 12 L. M. Burukm, Natunvtkenschafren,45 (1957) 293-294. 13 C. H. Yang and P. N. Srkasbv~ J. Reprod. Ferril., 37 (1974) 7-25. 14 B. A. B rermeseb D. P. Buhrer and A. A. Gottlieh, hrt& Biuchem., 87 (1978) 411-417. 15 H. Sternbach, R. Engehudt and A. G. L&us, Eb-. J. Biochem., 60 (1975) 51-55. 16 PzT. Rowley and J. Morris, Erp Cell Res., 45 (1967) 494497. 1 2 3 4

344

-

kkFARooQcJI

17 Fa F__CQX, Errr.1. Biochem. 39 (1973) 49-61. V_S_!wh.i*P_P&&RHeilar.KiW.Seif.&& 18 W_Zi?I&K_&hl,D-Rabassay.MM-!&hadnsa G&-f Se Ear&or Symp @zanZ~iYiof., 35 (1970) 47-58,

24 E. D. EEom,I. B&L &M., 215 (1955) l-14. 25 E. D. Kmn, I. Biol. C&m., 215 (1955) l!i-26. 26 R L. E&ten and c. Ix HolEenber& L t4rr;riRes.. 10 (1969) 374382. 27 T. F. W&yne, Jr.-and I. M. Fkk, C&c. Res.. 27 (1970) WI-951. 28 P. Iverius, V_ Lind&l, Ta Egehd and R. J_ OIiva-0~2, Biot. Ciiem.. 247 (1972) 6610-6616. 29 C. J. Fickiing B&&k Btopliys. Acru 280 (1972) 569-578. 30 K_ Jansen and W. C. Xulnnann, Bz%chih~~ Bioptrys.Artn, 369 (1974) 387-3%_ 31 C. & Ehnhofm, & Gre~en and W. V. Brown, &khik Biaphys. Acra, 360 (1974) 68-77. 32 DI3r. Lapp, B. We Patterson rind A. D. Ekeic, I. srbc. Ck, 246 (1971) 4557-4579. 33 k D. Ekein and M. M2&eU, Chkakyh. Rear.,37 (1974) 223-238, 34 A D. Elkin tid M. MitcJzeIl,Arch. i?khem Bioghy., 168 (1975) 369-377. 35 R T. Kuczen&i and A J- MandeZI, 1. Bi&_ &em.., 247 (1972) 3114-3122. 36 R T. Kuczmki and k J. Ma&e& 1. IV-_, 19 (1972) 131-137. 37 Ra T. Kuczms@ J. BzX Ckem.. 248 <1973)SOi4-SaSO. P_ H. Everiws,Bhchem. J., 124 (1971) 677-633. z R. Axes, J. Posth and S. Em&&, Narure (Londbn,l, 214 (1967) 1302-1304, 40 J. Porath, K- Aspbq, H. Dzvin and R Ax&, J, Ckromarogr., 86 (1973) 53-56. 218 (1968) 83+838. 41 J. Pora&, Narure (,h&m], 42 1. Danishefkky. Adwx. Exp. Med. siop., 52 (1974) 105-118. 43 Ps N. Sriwsava znd A. A. Famoqui, &hiem. J.. 183 (1979) 531-537. 44 M. Y. Vasshavskaya, A L. Klibanov, V. S. Gokkacher and V. P. Torchilh, AaaL B&&em_ 95 (1979) 44+451. 45 J. P. Hprnsn&, D_ 0. Anderson and C. PatM, J. Bloc. Ckem_, 233 (1958) 712-716_ 46 R- C. Drzeniek Noirae (.Luruf~~. 211(1966) 1205-1206. 47 G. DeIanbn&% G. We&r and A. Canteso, Amrr. J. Pkysfol.. 184 (1956) 415420. 48 T. A Bickk, V. Pirro&a and R. In&r, N&c A& Rec. 4 (1977) 2561-2572. 49 A. Bensadoun, C- Ehaho!m, D. Skinbsg and W. V. Brown, 1. Biol. Ckent.,249(1974) 222@2227. so P. & Iverius and A Mm ostlund-L&&pie, L BzoL Cketr~, 253 (1976) 77914795. 51 J. Boberg, J. Augush, M. L. Baginsky, P. Tejada and W. V. Brown. J. LiBfd Res., 18 (1977) 544-547. 52 A. 33asadoun and T. L_ Koh, J. Lx&3 Rec. 18 (1977) 7-773. 53 T. Kuusi, R. K J. Kirmunen snd C_ Ehnhoh, FEI3.SLcrr., 98 (1979) 314-318. 54 M. Tekzre, P. Penon, Y. Azon and J_ Ricard, FEBS Len., 82 (1977) 77-81. 55 J. A- Jachrhg, P. S. Woods and R G. Roedcr, J. WtoE Chem., 252 (1977) 8762-8771. 56 gF5_ Spindla, G. L. Dueser, J. Mn.Daksio and M. R. Faule, J. B&I. Ctian., 253 (1978) 466957 W. &lig, K. 0. Stetter and M. Tobien, E&r. L &&em.., 91 (1978) 193-199. 58 G. pflugfelderand Ja Sanabichkr, FEBS tit& 93 (1978) 361-364. 59 T_ E. Cawston and J- k Tyler, &&em_ A_. 183 (1979) 647-656_ 60 L_ H. Rome. I. Garvh and E. F. Neufefd, Arc&. B&&em B&J&S., 189 (1978) 344-353. 61 E Ekschkc and H. Kressc, Bhckem J., 181 (1979) 677-c&. 62 A. A. Faroaqui and P. N. Sriwsava, Fad. Proc., Fd tie Sot Eqx BLd.. 37 (1978) 1519. 63 L- EL Laarus and N. C. Kikon, Arch. &&em. BiopAgs 164 (1974) 414419. 64 G. Aderton, W. IF, Ward and HI_C_ J?kvoXd, A iakk C&m., 157 (1945) 43-58. 65 R.. !Zam!xwgh aid M. Smith. science, 186 (1974) 745-746. 66 J. Ehkh and S. S. Stivh, I, Phzrm. ,sci., 62 (1973) 517-544. 67 S. R Ss~~ P. L DoIan, B. Radhahrimamrnthy and G. S. Bererrson. Atkrawkvarfs, 16 (1972) 9sl&s.

PURIFICATION 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

OF EN2XMES

ON KEPm-KAROSE

345

Y. T. pan, A. we E=ruskiand A_ D_ EnJeiL&Arck gic-ehem.BiopFry_. 189 (f978) 231-24Q_ M. M. Anderscm, P. S. G&key and M. 3. segatchian, 2%mnb- Ztzs-, in press. M. M. Am, EL Borg and L. 0. Anderson. 2-hmz& R&s.. 5 (1974) 439-%52. F. A. Shelburne and S. H. Quarfordt. J. Chk. limsz., 60 (1977) 944-950. P. H. herius, J. B&T!.them, 247 (1972) 2607-2613. S. EL Qtu.&mB, R S. &in and L. Iakc& Biochem. BSphys. Rex. Conrnuar.,83 (2978) 786793. A. M. MO_ N. M&i%& 3. Mm nt and G. A. Puca, Roe- Nat. Ad. Sci., US. 74 (1977) 48&5-%90. F. Auricchio. k Rotor~di, P. SmnpaaIo and E. Schimme, Biochem.J., 171 (1978) 137-141. E. MuIder, A. Foekens, M. 3. Peters aad EL J. Vandermolen, FEBS LRtt., 97 (1979) 260-264_ P. Biamlki, BOX Sac. Ital. Bful. S&T.., 32 (1957) 1104-1109. CmEL Aborg arid B. Unms. Acte Pfrysiooz, Sand., 74 (1968) 552-557. A. A. Waldman, G. M&ix and 3. GoI&tein, Rat. Nat. Acad Sci. U.S., 72 (1975) 2352-2356. L. L Slabin. B&kern. Biophys. Res. Conmum., 73 (1976) 539-547. I. Kradce and 2. Dusek, &xfrtwz. J., 172 (1978) l-7. J. Hrsdee and 0. Krir. Biochcm. 3., 173 (1978) 349-352. J. A. Awad, P. J. Lupien, S. Moorjani and R. clontier, Cm. Ther. Res., 21 (1977) S&532. P. J. Lupicn, S. Mocxjani, M. Lou, D. Bmn and C. Game, P&t. Res_, 14 (1980) 113-117. T. Kahn and I. Owrton, J. Erp. Zuo., 171 (1960) 161-166. 0. Novo and k Dochan, Roe. Nat. Ad. S-d. U.S., 69 (1972) 2069-2076. E. F. Manmen, in N. U. Bang, F. EL Her, E. Deutsch and E. F. Maen (Editors), Thromhis rmd Bke&g Diwr&s, Academic Press, New York, London, 1971, pp. l-56.