A modified purification method and properties of pure porcine d -amino acid oxidase

ARCHIVES

OF

BIOCHEMISTRY

A Modified

AND

BIOPHYSICS

Purification

Method

D-Amino SHIAO-CHUN

TU,2 STUART

889-896

169,

and

Acid

(1973)

Properties

of Pure Porcine

Oxidase’

J. EDELSTEIN,

AND

DONALD

B. McCORMICII3

Molecular and Cell Biology, and the Graduate School of Nutrition, Cornell University, Savage Hall, Ithaca, New York i4850

Section of Biochemistry,

Received July 30, 1973 DEAE-Sephadex column chromatography now has been used for the final step in purification of n-amino acid oxidase apoenzyme. A specific enzymatic activity of 35-37 units/mg has been obtained for the pure holoenzyme. The purity has been established by disc and SDS gel electrophoreses and by sedimentation equilibrium. The molecular weight per enzyme monomer has been found to be 38,000 f 1000. Each enzyme monomer binds one FAD and one benzoate with dissociation constants at 23°C and pH 8.5 of 5.35 X 10e7M and 1.96 X 10e6M, respectively. The holoenzyme is more negatively charged than the apoenzyme at alkaline PH. The amino acid composition and some other physicochemical properties of the oxidase are reported.

The basic procedure for the purification of porcine n-amino acid oxidase [n-amino acid: 02 oxidoreductase (deaminating), EC dinucleotide 1.4.3.31, a flavin-adenine (FAD)-dependent enzyme, has been well established. The generally adopted purification met.hod can be summarized as follows: 1. Benzoate, a substrate-competitive inhibitor, is used as an enzyme stabilizer and the partially purified oxidase obtained as an iron-containing holoenzyme-benzoate complex after a series of heat t’reatment’s (1). 2. The removal of iron contamination and further purification of the oxidase is achieved with calcium phosphate column chromatography (2). 3. The iron-free holoenzymebenzoate complex is crystallized (2) or, alternatively, another heat treat’ment used (3). 4. Finally, the benzoate-free holocnzymc is obt’ained by repeated ammonium sulfate precipitation of the oxidase in the presence of an excess amount of substrate (4). * Supported in part by Kesearch Grant AM04585 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and in part by funds made available through the State University of New York. 2 Present address: The Biological Laboratories, Harvard University, Cambridge, Mass. 02138. 3 To whom reprint requests should be sent. 889 Copyright All rights

0 19i3 by Academic Press, of reprwluctim in any fwrn

Inr. reserved.

The crystalline n-amino acid oxidase so purified has a typical flavoprotein spectrum and usually shows but a single component upon ultracentrifugation (2). Nevertheless, discrepancies in amino acid composition and molecular weight of this enzyme have long been noted. The values for the content of neutral and acidic amino acids differ slightly in earlier reports, while quite different values for the content of basic amino acids have been assigned to this oxidasc (5-7). The molecular weight of monomeric oxidase, which binds one FAD, has been reported to be wi-ibhin the range of 45,000~55,000 (2, S-10). Recently, the monomeric oxidase has been reported to have a molecular weight of 37,000, based on sodium dodecyl sulfate (SDS) gel electrophorcsis (II), and 35,00040,000 shown by molecular sieve studies (12). The differences in t’hc reported molecular size and composit,ion arc beyond the range of usual experimental errors. This can be brst explained by the existence of impurities in the enzyme preparations. Herm and Ackrrs have demonstrat,cd that high mokcular weight, catalyt’ically inactive components could be removed from the apot’nzyme (12). Yagi et al. also reported thab t’he crystalline oxidase contained some impurities bascbd on the N-krminal analysis (10).

890

TU,

El)ELSTEIN,

wAmino acid oxidase has bwn one of the most extensively studied flavoproteins. Absolute purity of this cnzymr: has to bc cstablishcd before further work on the structure and function of the oxidaw should bc undcrtalon. WC dcmonxtratcd earlier that pure n-amino acid oxidasc could 1~1obtained by the use of preparative c+:ct~rophoresis (13). However, this method requires special apparatus and gives a poor yield. In this report we describe a simple modification of the purification method of ;\laswy et ab. (2). Follo~ving this modification, the apooxidase can be isolated in a form that is pure as shown by several physical criteria. W’o also present rcsults of studies on the molecular size! amino acid composition, and some ph\-sicochemical propertics of the pure enzyme. MATERIALS

8Kl)

hIETHOl)S

:l~ateriaZ.s. Guanidine hydrochloride (ultrapure) was purchased from Mann Research Laboratories. Riboflavin and 2,3,5-triphenyltetrazolium chloride were from Nutritional Biochemicals. Phenazinc methosulfate and FAI) (monosodium salt) were obtained from Eastman. I)E:.U-Sephadex (A-50) was from Pharmacia and was prepared for use by overnight treatment, with water and washing with saturated sodium pyrophosphate solution followed by 0.01 M pyrophosphate buffer, pH 8.3. All other reagents were of analytical quality from 11sua1 commercial sources. Glass-distilled water was used throughout the entire work. Assay of enzyme. The enzyme activity was measured either spectrophotometrically using n-phenylglycine as substrate (14) or by following the oxygen uptake with a Gilson model KM oxygraph, using D-alanine as substrate, as described before (13). In all cases, FBI) was maintained in the assay solution at 2 X lo-” Y to obtain the optimum activity. One unit of enzymatic activity is defined as 1 bmole of n-alanine oxidized to pyruvate/min at 38°C and pH 8.3. Protein concentrnt,ions were determined by the method of Lowry et al. (15). of enzyme b:y DEAE-Sephadex Ptcri$cation chromatography. Partially purified holoenzymebenzoate complex was first isolated from hog kidney following the method of Massey et al. (2) through the stage of calcium phosphate column chromatography. The benzoate was removed by repeated precipitation of the holoenzyme with ammonium sulfate in t,he presence of n-alanine (3,4). Apoenzyme was prepared from the partially purified holoenzyme by dialysis against KBr solution (1G) and was subsequently subjected to

AND

XIcCORMICK

DEAE-Sephadex (A-*50) colrunr~ chromatograph)~ for the final purification. (;rnerally, 30 -40 mg of partially purified apoenzyme in 4 ml of 0.01 M sodium pyrophosphate buffer, pH 8.3, were applied to a I)T
PURIFICATION

SNI)

PROPERTIES

taining 0.1 mg of apoenzyme in 0.2 ml of 0.09 u sodium pyrophosphate buffer (pH 8.3). Sedimentation equilibrium was reached overnight. ,4t, the end of the run, each sample was scanned for optical absorptions at 280 nm versus radial distances. Resrdts were printed orlt automat~ically in digital n,~mbers. RESULTS hrijcat~iotl~ of Bmynae by DldAE-Xephatle.~ C/I romafoy~apf~y. Partially purified apoenzyme was subjected to DEAE-Sephadex chromatography for the final purification as described in Methods. The clution pattern of a typical run is illustrated in Fig. 1. The cnzymatically actiw prot8cGn can be clearly separated from a minor inactive protein peak. The flow rate was great’ly ret’ardcd when t’hc clution buffer was changed to 0.1~ pyrophosphatc. For routine operations, the rlution was stopped after the act#ive fractions wre collectc~d. The extent’ and yield of t,h(l purification are summarized in Table I. The purity of enzyme preparations bcforc and after the DEUCSephadex chromat’og-

3 0.16

0 z 0.12 r \ 006

: $a

0.04

0

IO

20 30 40 FRACTION NUMBER

50

600

FIG. 1. Chromatography of partially purified apoenzyme on DEAE-Sephadex (&SO) column. Approximately 33 mg of apoprotein in 4 ml of 0.01 M pyrophosphate buffer (pH 8.3) were applied to a column. Conditions for chromatography were described in Methods. Protein concentration was measured by reading absorbances at 280 nm. Enzyme activity, expressed as aA 252/min, in 0.01.ml aliquots was followed at 23” C and pH 8.3 with o-phenylglycine as substrate (14). Concentrations of t)he elution buffer (pyrophosphate, pH 8.3) are indicated at the top portion of the figure.

OF D-.khIIN(l

ACID

OSIDAHE

891

raphy was &ted I\-ith analyt8ical gel ck+rc:phorews. The enzyme prepara,tion obtairwd from calcium phosphate column offlwnt could be diffcrentiakd, by bot’h disc and SDS gel elcctrophorcses, into a ma,~or slow-moving band and a minor fast’-moving band (Fig. 2, top). The major band was idt&ficd as the enzyme by st8aining for activity. Aft’w the subjcrted to same preparation has bm~ DEAFXephadex chromatography, t8hepurified enzyme oniy ckxistsas a single, :&ive protein component, as shown by disc and SDS gel electrophorcscs (Fig. 2, bot8tom). F’or comparison, the calcium phosphate column effluent was also subjwtcd to cithtr crystallization (2) or heat trc~at,mcnt~(3) for t8hoconv&ional final purificat’ion. With both prcparations, a diminished impurity band still exist’s aftclr analytical gel c~lwtrophorcsw (results not shown). Xolecular weight rlPte~lrzir,afiotcs. ‘l’h~ molecular w-eight of the apoenaymr purified by DEXE-Scphadcx chromatography \vas d(lt8crmined by SDS gel clcctrophorwis and sedimentation equilibrium. licsults of a single run of the molecular wight dctcrmination by SDS gel cktrophorwis are shown in E’ig. 3. An average of four such analgsw indicat8cLst’hat the purified n-amino acid oxidasc has a mol~~cular weight of 3S,OOOf 1000. The purifkd apornzymcx LVRHalso t’cated immcdiatcly aftc>r iscktion for molwular w-\-eightby scdimcntation clquilibrium as drscribed in ?tIethods. The results \vw(’ analyzcd by plotting t,he log of the optical absorbancc at “SO nm versus t,he square of the distance from the wntcr of the rot’or to the position whew the wspcctive protein concentration was measured. With t’riplicate samples, linear plot#s (correlation cocficients of 0.992, 0.994, and 0.995) wcr(’ obtained. This indicatcld thfl homogeneity of the enzyme preparations. Thr slopes were det.ermined from the plots bawd on least-squares analysis. The molecular weight was calculatcd to be 37,600 f 1700 from thr slopes and using a parCal specific volume of 0.740 cm3/g. The partial specific volume was determined from the amino acid composition of t’he purified n-amino acid oxidasc (Table II), assuming equal distribution of the amide nitrogrn

892

TU,

EDELSTEIN,

AND TABLE

F%JMMARYOF PURIFI(I.\TION

OF

D-AMINO

Acre

MCCORMICK I

APOOSID.\BE WITH I~~!M~-%~~IoE~

COLUMN

CHI~OM.ITO~I~~PIIT

Apoenzyme t phosphate Concentrate Sephadex

4.0

33.0

841

2.5

5.4

17.3

631

3G..i

between glutaminc and asparagine, by the method of Cohn and Edsall (22). Amino acid composition. The purified namino acid oxidase was hydrolyzed in 6 N HCl for 24, 48, and 72 hr as described in Methods. The average or extrapolated value of each amino acid residue n-as calculated according to the method of Tristram and Smith (23). The amino acid composition of a monomeric enzyme unit, taking 3S,OOOas the molecular weight, is summarized in Table II. The oxidase has a high cont’ent of aromatic amino acids. The absorbance at 280 run of a 0.1% apoenzyme solut’ion has been found to I)(,, ‘)-.i.‘I’, . Hinclinq of FAD. Apoenzymt3 was tikatcd \rith FAD solutions and t’hc intensity of protjck fluorrscence at different FAD concentrations was followed (insert, of Fig. 4). The rcwlt,s were analyzed by plot’ting (12 - l)(FAD)o versus

as shown in Fig. 4 according to the method of Kurganov et al. (24). Symbols are: (FAD), , molar concentrat’ion of Ootal FAD (free plus enzyme-bound) ; 10 , fluorescence intensity in the absence of FAD; I and I’, fluorescence intensity at total FAD concentration of (FAD)o and (FAD),,&, rcspcctivcly; and k, a preset constant multiplier. Least-squares analysis was used for the linear

Yield (‘Z 1

Specific activity (units/mg protein)

(ml)

VOlLlIlW

after calcium column from l>EARcolumn

Total activity (units)

Total protein (W

Total

Stage

Purification (-fold)

1

101)

.i

1.43

73

0

5s

$ a

a I 2

0

0.3

0.6

0

03

0.6

0

0.3

0.6

0

MOBILITY

Fro. 2. Electrophoretograms of n-amino acid oxidase before (top) and after (bottom) DEAESephadex chromatography. Approximately 20 fig of prot,ein per gel were used for disc gel electrophoresis (columns A and B) and 10 rg protein per gel were used for SI)S gel eleckophoresis (column C). Enzymes were electrophoresed either as apoprotein (columns A and C) or as holoenzyme (column B) and were stained for protein (solid lines) and activity (dotted lines) as described in Methods. Gels were scanned at G30 nm for the protein stains and 510 nm for the activity stains.

fitting. The value of I,/10 and (fi), can be determined from the intercepts of such plot on both axes where 1, is the fluorescancr intensity at (FAD)0 + m and (E)” is the total molar concentration of FAD-binding site. Following such analysis, the (fi), was found to bc 4.93 X 1OV .\I for a 0.02% apoenzyme solution. Such an c’nzymc> solution has a molar concentration of 5.26 X 1OP nr based on 38,000 RIW for a monomeric (‘nayme unit. Thus, the number of PAD-binding sites per monomeric unit can be calculated to bo 0.93,

PURIFICATION

ANT)

PROPERTIES

OF u-AMINO

ACII)

TABLE AhuNo

ACID

rlmino acid

MOBILITY FIG. 3. Molecular weight determination of I)amino acid oxidase by SI)S gel electrophoresis. The standard curve was constructed with the following protein standards (0) of decreasing molecldar weights: serum albumin, catalase, yglobulin (heavy chain), liver alcohol dehydrogenase, pepsin, trypsin, and ribonuclease. The molecular weight of the apoenzyme of o-amino acid oxidase (0), purified by l)EAE-Sephadex was done according to the chromatography, method of Weber and Osborn (11).

which suggests that each enzyme monomer binds one FAD. The dissociation constant for the enzyme-FL4D int’rraction (KdFiiss)is defined as KF.

= (E’)(FAD) (Es FAD)

dlss

where (E), (FAD), and (E .FAD) are cquilibrium molar concentrat’ions of the free enzyme monomer, free FAD, and enzymeFAD complex, rc>spectively. The KIiiss can then bc calculated, at different, conccntrat,ions of FAD, by the following equation (24) : (t-?)[(l-k)

.(FAD)o - (1 - ;) K&s

(El,]

= (l-:0)(1

+)

*

The mean value of the dissociation constant has been found to bc 5.35 X 1O-7 11at 23°C and pH 8.5. Binding of Demoate. The substrate-binding property of n-amino acid oxidase was PX-

OF D-AMINO

Nearest integer of residues/ 38,000 g protein

14.0” 9.0 37 .s 20 .i 31..i 21 .Y 12.3” 36.4 22.1 31.7 lG.7 2F.V 4.G” 18.4C 35 G 13.1 14.2 8.(id 4.r,”

CJ3

‘I Calculated

II

COMPOSITION ACID OXID.\SE

Residues/ 38,000 g proteinI‘

Lys His NH, Arg Bsx Thr Ser Glx Pro Gly Bla Val Met He Leu Tyr Phe Tw

893

0SIl)ASE

according

to Triskam

14 !I 38 21 32 22 12 36 22 32 17 27 5 18 36 13 14 .7

and Smith

(23). b Value obtained by extrapolation to zero time. c Value at 72.hr hydrolysis. ci ljetermined calorimetrically as described in Met hods.

amined by t’itrating the holocnzgme with bcnzoatc, a substrate-compet8itive inhibitor. Absorpt!ion difference spectra (enzyme-benzoat’c complex minus holocnzymc) sho\v a peak at 300 nm and 110 changes above 533 nm (resulk not shown). The difference absorbanw (ALA) at 500 nm was thrn followd to assess the binding of benzoate to enzyme (iusrrt of ITig. 5). The met,hod of Wu a,nd Hammcs (25) was adopted for t’hc dctcrminat8ion of the number of bcnzoatc-binding sites prr enzyme monomer, II, and thcl dissociaGon con&ant for the c>nzymct-bcnzoate interaction, K& . Thr lattrr is defined as &$,,

= [(fi)o

- I-‘][(Benzoatc)o P

- Q]

>

where (E), and (Brnzoatc)o are thca molar concentrat’ions of total cnzymc monomer (3,000 1\IW) and total benzoatc, rcspwtively, and P is thr concrntration of enzyme

s94

TU, 24

I

I

I

20 -

EDELSTEIN, I

10

AND

MCCORMICK

complexed with benzoate. The above equation can be rearranged to give

I

08,

(Bcnzoatc)O/P

Bk=2

4-

(0)

k = 3

(m)

k =4

(A)

0

I

I

I

I

0.2

0.4

0.6

0.8

1.0

1.2

FIG. 4. The fluorescence quenching of n-amino acid oxidase with FAD, and graphical determination of the dissociation constant and number of FAD-binding sites. A 2-ml pyrophosphate solution (0.05 M, pH 8.5) containing 0.4 mg of apoenzyme was titrated with microliter increments of FAD solutions at 23°C. Fluorescence intensit,y was measured at 330 nm with excitation light at 280 nm.

I

I

I

I

I

a\ 0 iYi 2 3 5 m

T .-

I

,

I I

I

I

2

3

CBENZOATE) I

4

105.1 HI A

5

6

I/C(E),-PI(IO-5~M-') FIN;. 5. Titration of holoenzyme with benzoate and graphical determinat,ion of the dissociation constant and number of benzoate-binding sites. A l-ml pyrophosphate solution (0.1 M, pH 8.5) containing 0.8 mg of apoenzyme and 10e4 M FAD was’titrated with microliter increments of benzoate solut,ions at 23’C. The difference absorbance at 500 nm was followed with a Perkin-Elmer Two-Wavelength Double-Beam Spectrophotometer 3.X using 540 nm as t,he reference light.

= K,Bi,,/[(E)o

-

P] + n.

The term 1’ can be calculated at different benzoatc concentrat’ions by the equation P = (AA/AA,,,)(E)o , where AA,,,,, is the diffcrence absorbance at 500 nm when the enzyme is completely saturated with benzoate. I’ollowing such analysis, a plot of (Benzoatc),,,/P versus l/[(E)0 - P] has a slope equal to Kiisa and an intercept equal to 11(25). From least-squares analysis of the results presented in Fig. 5, 11and Kfiss for t’he binding of benzoate by n-amino acid oxidasc have been found t)o be 0.9s and 1.96 X low6 31,respcctively. The observed stoichiometry of bcnzoate binding suggests that one enzyme monomer (38,000 R’IW) binds one benzoatc. DISCUSSION

For the purification of porcine n-amino acid oxidase, the method of Massey et al. (2) was followed through the stage of calcium phosphate column chromatography. The cnzyme obtained ran be clearly shown to contain an inactive protein component by analytical gel clcctrophorcscs. The molecular size of the inactive protein component (rstimated to be approx 34,000) is only slightly smaller than the oxidase based on SDS gel elect,rophorctical behavior (Fig. 2, top C). Attempt’s for the separation of the impurity from the oxidase by molecular sicvc chromatography with Sephadex G-100 and G-150 have been made but wit’hout success. Sevcrt#heless,this inact’ive component moves faster than both holo- and apoenzynws t’oward the anode on disc gels (I?ig. 2, top A and B). Such differences in mobility must he mainly due to a charge effect, since t’he molecular size of t’he impurity differs only slightly from the oxidase. It is int’eresting to n0t.e that, apocnxyme is less negatively charged than the holoenzyme at pH 9.5 (operation pH of clcctrophorcsis) and mn be separat’cd bcttcr from the impurity on disc gels. Based on these observat#ions, DINE-Sephadex column chromatography was devised for the scparation of apocnzymc from impurity by charge differences. The homogeneity of the enzyme purified by such an operation is establishhcd

PURIFICATION

AND

PROPERTIES TABLE

PHYSICOCHEMICU

Physical parameter Molecular

weight

Partial specific A&t% FAD-binding KL Benxoate-binding

volume

PROPERTIES

OF n-.aIINO

ACID

III OF D-AMINO

ARID

OYIDASE

Value

Method SDS gel elect,rophoresis Sedimentation equilibrium lmino acid composition Optical absorption Titration with FAT)

38,000 37,600 0.i40 cm3/g 2.25 0.93 FAD-binding site per 38,000 h;IW .j.3,5 X lo-’ M at 23”C, pH 8.5 0.98 Benzoate-binding site per 38,000 MW 1.96 x lO-6 1~ at 23”C, pH 8.5

by disc gel electrophoresis, SDS gel elect’roequilibrium. phoresis, and sedimentation Moreover, a specific enzymatic act’ivity of 35-37 units/mg has been obtained with t’he purified enzyme which is considerably higher than earlier reported values ranging from 17 to 29 units/mg (3, 26). The presently observed inactive protein component is obviously different from the high molecular weight inactive species observed earlier by Henn and Ackers (1‘2). WC believc that this high molecular wight component is most likely a protein polymer dcrived photochcmically from t’he native Damino acid oxidase (13). No such high II-IOlecular weight component can be dctcct’rd if the enzyme preparat#ion is carefully shicllded from light during the course of purification and analysis. The molecular weight and amino acid coralposition wit,h clarlier preparations of I)amino acid oxidaw have long been a mattcxr of inconsistency. With t,he present apparcnt’ly pure apocnzyme, the molecular weight of enzyme monomer has bcrn shown t#o be 38,000 f 1000 based 011 SDS gel electrophoresis and sedimentation equilibrium and is in good agreement, with the molecular weight per FAD-binding sit,c and per bcnzoatc:-binding site. The amino acid composition of the pure apoenzyme has been drtcrmined and presented in Table II as rcsidws per monomeric unit with molcculwr weight of 38,000. Some other physicochemical properties of the pure n-amino acid oxidase have also been

595

OSIDASE

Titration Titr:tt,ion

with with

FBD benzoate

Titration

with

benzoate

examined, and all results are summarized in Table III. REFERENCES 1. KUBO,

H.,

W.\TIRI, S.\\V.U)A,

Y.\M\No, N, IJVATSUUO, SOYAM~, T., SHIR~ISHI, s., K.Lw.\sHIM.\, N., iLIIT.\NI,

G-D

K.

ITO,

H.,

(1958) &ill.

sot.

Chim.

bl.,

J., S.,

Biol.

40,431. C., .JND BENNGTT, R. 2. LU.G~EX--, V., P.\LMER, (1961) Biochim. Uiophys. Ada 48, 1. P. E. -LSD L~.\YsEY, V. (1968) 3. BRUMB~, Biochem. Prep. 12, 29. 4. YAGI, K. .\XD Oz 1w.1, T. (1962) Biochim. Biophys. Acta 66, 420. A., H.LR.\D.~, N., .\ND YAGI, K. (1967) 5. KOT~KI, J. Biochent. 61, 598. I,. .\ND CUFFED, T). S. (1967) 0. HEIJ,EILMIN, J. Biol. Chem. 242, 582. B. 11. (1969) i. FOND.\, bl. I,. .\.TD ANDERSON, J. Biol. Chem. 244, 666. B. M. (19G8) 8. FOND.\, hl. I,. .\ND AKDERSON, 1. Biol. Chem. 243, 5635. 9. ?~IIB.LKE, Y., ABE, T., :\ND Y~M.LNO, T. (1971) .I. Biochem. 70, i19. 10. Y.\cr, Ii., NAOI, M., H:\R.LD.~, M., OIC.\MCRA, T., .LND KOTIKI, K., 1111,\I<.\, II., OZAWI, A. (1967) J. Biochern. 61, 580. 11. WEDER, K. .LND OS~OI~N, bl. (1969) 1. Biol. Chcm. 244, 4406. 12. HEXJX, S. W. BND ACKERS, G. K. (1969) J. Bio2. Chem. 244, 465. I). B. (1973) J. 13. TLT, S.-C. .~ND ~IcCort~rc~~, Biol. Chem. 248, 6339. B. M. (1967) 14. FONDA, nl. L. .\SD ANDERSON, J. Biol. Chem. 242, 3957. N. J., F.~ILR, 15. Lo\\.RT, 0. H., ROSEBE~~G~, h. L., AND R \ND.\LI>, R. J. (1951) -1. Biol. Chcm. 193, 267. V. .\NI) CURTI, B. (1966) J. Viol. IG. XUSE:U, Chem. 241, 3417.

S9G

TU,

EDELSTEIN,

0. (1971) d/ethods 17. GABRIEL, 565. 18. HAYES, M. B. AND WELLNER, Hiol. Chem. 244, 6636. 19. SPIES,

J.R.

CHAMBERS,

D. D.

22,

(1969) J. C.

(1949)

Chem. 21, 1249. G. L. (1959) Arch. Biochem. Biophys.

Anal. 20. ELLMAN,

82, 70. 21. YPHANTIS, 22. COIIN,

AND

Enzymol.

AND

E.

1). A. (1964) Biochemistry 3, 297. J., .ANLI EDFULL, J. T. (1943) Pro-

McCOltMICK

teins, Amino Acids and Peptides, p. 370, Reinhold, New York. 23. TIUSTILQX, G. It. .\NLI SMITH, R. H. (1963) Adv. Protein Chem. 18, 277. 24. KUR~.LNOV, B. T., Sunrto~~v.~, N. I’., 1x1) Y.II~~VI,EV, \?. A. (1972) FEBS /,efl. 19, 308. 2.j. Wu, C.-W. .\xI) H.\MMEB, (+. (+. (1973) Uiorhemisfry 12, 1400. L., COFFEY, I). S., AND NEIMS, 26. HEI,LERM.\N, A. H. (1963) J. Niol. Chem. 240, 290.