Aminoethylation and fractionation of glutenin evidence of differences from gliadin

404

BIOCHIMICA ET BIOPHYSICA ACTA

~BA 3525 o AMINOETHYLATION AND FRACTIONATION OF GLUTENIN EVIDENCE OF D I F F E R E N C E S FROM GLIADIN*

J O H N A. R O T H F U S ANn M. J. A. C R O W

Northern Regional Research Laboratory*', Peoria, Ill. 6x6o 4 (U.S.A.) (Received March i2th, I968)

SUMMARY

After reduction and aminoethylation, glutenin can be separated into four groups of proteins by fractional precipitation from 0.5 M acetic acid solution with Cu(N03) 2. Material precipitated by 0.0283 M Cu(N03) 2 accounts for 22% (w/w) of the glutenin and consists primarily of slow moving electrophoretic components. This fraction, which has an amino acid composition unlike other proteins from gluten, contains large amounts of glycine and tyrosine and low levels of histidine, methionine, valine and cysteine. The remaining fractions from glutenin resemble other gluten proteins more closely in composition. Gel permeation chromatography of the protein components from glutenin shows they have higher apparent molecular weights than gliadin.

INTRODUCTION

Wheat gluten, the protein complex responsible for the unique cohesive-elastic properties of bread dough 1, can be separated into an alcohol-soluble fraction, gliadin, and an alcohol-insoluble fraction, glutenin. These two groups of proteins have similar amino acid compositions ~ but differ substantially in physical properties. Whereas purified gliadin proteins appear to have molecular weights near 2o ooo by both ultracentrifugal analysis a and amino acid composition s, the molecular weights of glutenin components range into the millions 5. Glutenin also has significantly higher solution viscosities than gliadin 5. In part, the unique characteristics of glutenin appear due to the presence of intermolecular disulfide bonds. Reagents that cleave disulfides reduce the molecular weight of glutenin to about 2o ooo (ref. 5), cause a sharp drop in viscosity of glutenin solutions, and produce material that behaves more like gliadin upon electrophoresis 6 * A preliminary r e p o r t of this work was presented at the i 5 5 t h National Meeting of the American Chemical Society, San Francisco, Calif. U.S.A., March, 1968. ** This is a l a b o r a t o r y of the N o r t h e r n Utilization Research and D e v e l o p m e n t Division, Agricultural Research Service, U.S. D e p a r t m e n t of Agriculture.

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AMINOETHYLATION AND FRACTIONATION OF GLUTENIN

405

in starch gel. The similarity of reduced glutenin to gliadin prompted WOYCHIK et al. 6 to suggest that glutenin might arise from gliadin-like proteins. Subsequently, BECKWITH and coworkers, in studying the reversible reduction and oxidation of gliadin 7 and of glutenin 8, noted that reduced gliadin could be reoxidized more easily than reduced glutenin, and that the reoxidized-reduced proteins differed in electrophoretic mobility and solubility. From these differences, BECKWlTH AND WALLs inferred that the structures of proteins which make up glutenin are probably different from those of gliadin proteins. Direct chemical comparison of individual proteins from glutenin and gliadin is complicated, unfortunately, by the properties that make these proteins useful. In the case of glutenin, limited solubility, cohesiveness, and a tendency to form intermolecular disulfide bonds make resolution of the unmodified proteins especially difficult. This paper describes use of the S-aminoethylation method developed by RAFTERY AND COLE9 to obtain derivatives of glutenin proteins that are more amenable to fractionation. Analysis and chromatography of the partially resolved aminoethylglutenin proteins give further data pertinent to the question of the relationship between glutenin and gliadin. MATERIALS AND METHODS

Glutenin and gliadin were obtained from hard red winter wheat flour by the method of JONES et al. 1°. Aminoethylation was carried out as follows : Glutenin (2 g) was dissolved in 200 ml of a solution containing 96 g of urea, 20 mg of disodium ethylenediaminetetraacetic acid, and 4 ° ml of ammediol buffer (4 M 2-amino-2-methyl1,3-propanediol, adjusted to pH 8.6 with dilute HC1) in a 5oo-ml glass-stoppered erlenmeyer flask. Nitrogen gas was bubbled through the solution for 0.5 h, i ml of mercaptoethanol was added, the flask was stoppered, and the contents were stirred slowly on a magnetic stirrer for 20 h at room temperature. At the end of this time, 4 ml of ethyleneimine was added in three portions over a period of 0. 5 h, and stirring was continued until the reaction mixture gave a negative nitroprusside test for sulthydryl (about 40 rain). The reaction was stopped by addition of 15 ml of glacial acetic acid, and the solution was dialyzed exhaustively against o.oi M acetic acid, and then lyophilized. Aminoethylation of gliadin required higher concentrations of mercaptoethanol (0.69 ml/g protein) and ethyleneimine (2.75 ml/g protein.) The preparation of S-cyanoethylglutenin is described elsewhere 11. Salt fractionation of S-aminoethylglutenin was conducted as follows: A solution (IO ml) of S-aminoethylglutenin in 0.5 M acetic acid was fractionated by addition of o.I-ml portions of 0. 5 M Cu(NOa) 2 at room temperature. At three different times, after the addition of a total of 0.6 ml, 2.0 ml, and 5.0 ml of Cu(NQ)2, the mixture was centrifuged in an International* clinical centrifuge (head No. 804) at 2500 rev./min for I h at room temperature to remove precipitated proteins. The precipitates, dissolved in 0.5 M acetic acid, and the final supernatant solution were dialyzed thoroughly against 0.5 M acetic acid, and then lyophilized. Recoveries were calculated on a dry weight basis. S-Aminoethylcysteine was prepared by a modification of the method of CAVAL* The m e n t i o n of firm n a m e s or t r a d e p r o d u c t s does n o t i m p l y t h a t t h e y are r e c o m m e n d e d b y t h e D e p a r t m e n t of A g r i c u l t u r e o v e r o t h e r firms or s i m i l a r p r o d u c t s n o t m e n t i o n e d .

Biochirn. Biophys. /lcta, i51 (I968) 404 412

•~-()(1

.I.A. ROTHFUS, M, j. A. CROW

H N I et al. 12. Cysteine hydrochloride monohydrate (I7. 5 g) dissolved in 200 ml of boiled water was taken to pH 8.5 with concentrated NH a and immediately reacted with 0.2 ml of ethyleneimine. The pH was maintained by bubbling CO2 into the solution, and the reaction was allowed to proceed with stirring at room temperature. After I h, pH was adjusted to 5.o with HC1, and then the reaction mixture was concentrated to 45 ml on a rotary evaporator. Dilution of the concentrated reaction mixture with 5 ° ml of ethyl alcohol precipitated i i g of crude aminoethylcysteine, which was separated by filtration and washed, first, with cold 5o% ethylalcohol and then with acetone. An additional 2.4 g of product resulted from further concentration of the mother liquor. The combined precipitates were recrystallized from alcohol water acetone as described by CAVALLINI8t al. 12 to yield IO g of ninhydrin-positive crystalline material. M.p., 196 x97 ° d. (Kofler) ; 197.6 ° d. (Mettler F P - I automatic melting point apparatus). On the amino acid analyzer, this material gave a single peak which emerged between lysine and histidine, as expected of S-aminoethylcysteine. Amino acid analyses were conducted as described by SPACKMAN et al. la on a Phoenix Model K-8ooo analyzer, modified for accelerated operation. A o. 9 cm × 15 cm column was substituted for the o.6 cm × IO cm column normally used to separate basic amino acids. An Infotronics (Houston, Texas) integrator and an IBM II3O computer provided automatic computation of amino acid analysis results 14. Electrophoresis of proteins in starch gel was carried out in o.o25 M aluminum lactate-3 M urea buffer (pH 3.1) according to the method of WOYCHIK et al. 1'5 as modified by BECKWlTH and coworkers a6. The cystine content of glutenin was measured directly by amperometric titration of the sulfite-treated iv protein by the method of BENESCH, LARDY AND BENESCHas with modified ~9 apparatus after ROSENBERG, PERRONE AND I{IRK2°. Sephadex G-zoo (Pharmacia) used in gel permeation chromatography was expanded in water and freed of fines by decanting any material still suspended after standing 15 rain. This sizing procedure was continued until the supernatant was clear, and then it was repeated in 0.5 M acetic acid. Chromatographic columns (3.8 c m x 155 cm and 1. 9 cm × 15o cm) were poured from slurries of the Sephadex in 0. 5 M acetic acid. Chromatography was also carried out in 0.5 M acetic acid. Column flow rates were 29 ml/h for the larger diameter column and 5 ml/h for the smaller. Colunm effluents were collected in 5 Io-ml fractions, and their optical densities were measured at 280 m# with a Beckman DU spectrophotometer. Effluent corresponding to each absorbance peak.was combined and lyophilized before further use. Sample recoveries were calculated on a dry weight basis. RESULTS

S-A mino-ethyl-glutenin

Studies by SELA and associates ~1 on polypeptidyl gliadins and by BECKWITH, WALL AND DIMLER22 on gluten derivatives, in which amide groups were hydrolyzed or converted to esters, showed that the solubility of gluten components can be improved by either introduction of additional negative groups into the molecules or disruption, in other ways, of hydrogen bonding by side-chain amides. S-Aminoethylation of reduced glutenin proteins is a relatively mild means of improving their solubility. Whereas both glutenin and derivatives which contain uncharged cyanoethyl groups Biochim. Biophys. Acta, i6o (I908) 4o4-412

407

AMINOETHYLATION AND FRACTIONATION OF GLUTENIN

are only sparingly soluble in water or dilute acetic acid, aminoethylglutenin was readily soluble at 5 % in 0.5 M acetic acid and even dissolved slowly at o.oi % in o.ooi M sodium acetate. The additional charged groups produced by aminoethylation markedly increased the electrophoretic mobilities of most of the proteins. Fig. I compares the starch-gel electrophoresis pattern of aminoethylglutenin with that of cyanoethylglutenin, which is representative of reduced glutenin 6. The unmodified glutenin from which these derivatives were prepared remained at the origin during electrophoresis. Presumably, if increased positive charge were the only change produced by aminoethylation, and if the same number of amino groups were introduced into each protein, bands in the aminoethylglutenin pattern should bear the same relationship to each other as do bands in the cyanoethylglutenin pattern. Although the patterns of both derivatives had the same general form, there were enough differences between the patterns to suggest that some proteins were modified more than others and thus might contain higher concentrations of cystine. The fl-region in aminoethylglutenin, for example, consisted of a relatively intense band followed by less prominent bands, but in the /~-region of cyanoethylglutenin the major component appeared in a trailing position. In addition, the aminoethylglutenin pattern contained extra fast moving and slow moving bands.

e

q

........

+

61utomim

A

I ....

t

++ ++ |+

. . . . . . . .

Fig. I. Starch gel electrophoresis of aminoethyl (AE)+ and cyanoethyl (CN)-glutenins. I~'ig. 2. Starch-gel electrophoresis of aminoethylglutenin and its fractions from salt precipitation.

Partial fractionation of arninoethylglutenin Fig. 2 shows the electrophoretic patterns of fractions after salt precipitation of aminoethylglutenin with Cu(NOa) 2. Distribution of sample and conditions of fractionation are summarized in Table I. Precipitation of proteins that gave slower moving electrophoretic bands (Fraction A) was nearly complete at a cupric ion concentration of 0.0283 M. This fraction, which accounted for 22% of the dry weight of the sample. Biochim. Biophys. ,4cta, 151 (t968) 4o4-412

4o~

j.A.

ROTHFUS, M. J. A. CROW

TABLE l DISTRIBUTION OF AMINOETHYLGLUTEN1N AFTER SALT PRECIPITATION WITH (~u(NO3) 2 Fraction

° o Recovered"

C~" ~ (31)

pH

A B C D

2t. 7 64.8 6.8 6. 7

0.0283 3.2 0.0833 o.1667 o.I 667"** 1.8

Relative ease of solution** + + @@+ + +--++

* C a l c u l a t e d on d r y w e i g h t basis ** Q u a l i t a t i v e e s t i m a t e : F r a c t i o n A, l e a s t r e a d i l y soluble in 0. 5 M a c e t i c acid. **" S u p e r n a t a n t from F r a c t i o n C.

best retained the descriptive properties of intact glutenin. About 6 5 % of the sample was recovered in a fraction that contained most of the fl-aminoethylglutenin proteins along with some faster moving components (Fraction B). The final 13% of tile sample was equally divided between two fractions, one containing mostly a- and some flaminoethylglutenins (Fraction C), the other consisting of proteins that remained in solution (Fraction D). Whereas the separations of Fraction A from B and Fraction C from D were each marked by distinct changes in the character of the precipitation, the separation of Fraction B from C was less clear cut. The ionic strength at which most of the fl-aminoethylglutenin proteins were confined to Fraction B was established by finer fractionation of the a- and /5-aminoethylglutenin proteins with smaller aliquants of Cu(NO3)2 solution. Other variables such as pH, protein concentration, or solvent dielectric constant have yet to be thoroughly explored as means of improving the bulk separation of aminoethylglutenin proteins. Precipitation with Cu(NOa) 2 was advantageous in that it produced comparatively well-defined fractions and could be duplicated easily. Similar, though less wellresolved, fractions were also precipitated with NaC1, NaNOa, FeC13, Fe(NO3)8, AgNO3, Hg(NO3) 2, and Cr(NO3) 3 at the same ionic strengths.

Amino acid composition of aminoethylgluteninfractions Table II compares the amino acid compositions of glutenin, aminoethylglutenin, and fractions from aminoethylglutenin. These analyses were obtained with samples that, in most instances, had been hydrolyzed only 20 h and are uncorrected for changes that might have occurred during hydrolysis. Except for valine, the values for our glutenin preparation are generally consistent with published data on glutenin 2. Our values for half-cystine and aminoethylcysteine in glutenin and aminoethylglutenin agree favorably with determinations by BECKWITH AND W A L L s, who found glutenin to contain 12.8 moles of half-cystine per lO5 g of protein. Comparison of the analyses of glutenin and aminoethylglutenin shows that the cystine residues were quantitatively derivatized by reaction of the reduced protein with ethyleneimine. Aminoethylation produced little or no change in levels of the other amino acids. In contrast to the general agreement of analytical data on whole glutenin and alkylated glutenin, differences between the fractions of aminoethylglutenin are quite striking. Fraction A was significantly different in content of aspartic acid, glutamic Biochim. Biophys. Acla, 16o (t968) to4 412

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TABLE II AMINO ACID ANALYSES OF FRACTIONS FROM GLUTENIN A m i n o acid

Millimoles of amino acid per zoo g protein* Glulenin

Lys His Arg Asp Thr Ser Glu Pro Gly Ala

ii.i 11. 5 20. 9 18.3 25.7 59-3 294.8 116.8 74.0 28.6

Cys~ Val

Met [le Leu Tyr Phe AminoethylCys % Sample weight recovered as amino acids

i3.5 t 29.o lO.5 23.0 53.0 24. 7 25.5

Amino-

Fraction

ethylglutenin

A *"

IO.O lO. 5 20.6 17. 5 25.0 57.3 289.2 lO9.7 71.3 26. 4

7.4 8-7 3.7 12.5 8-3 5.2 13.1 5.1 15. 9 lO. 4 16.9 23. 3 16.2 22. 4 21. 4 8.9 20. 4 20. 4 19.6 17.8 25.1 25.0 22. 3 22.2 24. 7 58.8 60.0 46.0 42.1 57-5 346,9 280.0 258. 9 246.4 290.9 128, 5 i i i . o 119.8 114.6 115.8 126.9 59.5 29.0 33.7 70-3 22.1 28. 3 25. 7 25. 7 26.6

. . 28.2 8.5 23.2 52.1 24. 7 25-5 12. 7

94

91

.

B**

. 9.5 2.9 12.9 37.4 42.9 18.6

. 33-9 IO.i 26.1 56.9 2o.1 29.0

4.6

I4.2

97

98

C

D

. . 38.2 32.I 9.2 lO.6 31.3 25. 3 54-7 44.9 6.0 8.8 31.o 26.8 3.8

82

11.8

A m i n o acid confetti recovered in fractions A-D ***

28.8 8.6 23.5 51.7 23.3 26. 7 11.2

85

* Corrected for moisture content. "* Corrected for ash. *'* Represents total of amino acids in Fractions A - D , weighted according to percent (w/w) of sample recovered. * Determined by amperometric titration.

acid, glycine, valine, methionine, isoleucine, and tyrosine. Ratios ranged from o.25 for valine (Fraction A/Fraction C) to 4.4 for glycine (Fraction A/Fraction C). Fraction A also differed markedly from low molecular weight glutenin 16, gliadin2,16, a-gliadin 23, y-gliadins 4, and other protein preparations from gluten 24. Fractions B - D , which were generally more similar, resembled a- and fl-gliadin. Compared to the other proteins, Fraction A was especially rich in glycine and tyrosine and poor in histidine, methionine, valine, and cysteine. Fraction C was also comparatively deficient in histidine and eysteine.

Gel permeation chromatographyof fractions from aminoethylglutenin To gain an estimate of their molecular size, the aminoethylglutenin proteins were compared to gliadin by gel permeation chromatography. BECKWITH and coworkers le showed that the gliadin fraction from wheat consists of proteins that have molecular weights near 30 ooo and IOO ooo. Thus characterized as a mixture of different molecular species, crude gliadin is a useful standard for comparison to aminoethylBiochim. Biophys. Acla, 151 (1968) 4o4-412

41o

J.A. ROTHFUS, M. J. A. ('ROW

~

A I

~0.~ co ,,; 0.: ~=0.:

[

lO0

200 300 Volume, ml

I

400

) Volume, ml

Fig. 3. C o l u m n c h r o m a t o g r a p h y of gliadin (A) a n d a m i n o e t h y l g l u t e n i n (B) on S e p h a d e x G-IOO ( c o l u m n 1. 9 c m X 15o c a ) in o.5 M acetic acid (i ml of 5~o solution). H e a v y lines on t h e abscissa indicate c o m b i n e d fractions. B a r g r a p h s below t h e elution p a t t e r n s indicate w e i g h t d i s t r i b u t i o n , c a l c u l a t e d on a d r y w e i g h t basis.

glutenin proteins. Fig. 3 shows elution patterns and weight distributions resulting when gliadin and aminoethylglutenin were chromatographed on Sephadex G-Ioo. Whereas the largest portion of gliadin emerged at V e / V o ~ 1.86, as expected for molecules with molecular weights near 3 ° ooo (ref. 25), the higher molecular weight components were hardly retarded on the gel (Ve/Vo = 1.o9). Purified gliadin 1~ which had been reduced and aminoethylated gave a V e / V o value of 1.85. In comparison, V e / V o ratios for Peaks I - I I I of aminoethylglutenin were 1.o 9, 1.23, and 1.63, respectively. These values correspond to apparent molecular weights of more than ioo ooo for Peak I and approx. 8o ooo and 4o ooo for Peaks I I and I I I , respectively. Similar results were obtained by CROW AND ROTH~US11 upon chromatography of cyanoethylglutenin on Bio-Gel P-3oo in 8 M urea.

6°°

Volume, ml

~00

,00, ,00

,;0

Volume, ml

~0~6 ,,6

Fig. 4. C o l u m n c h r o m a t o g r a p h y of F r a c t i o n A (A) a n d F r a c t i o n B (B) f r o m t h e salt p r e c i p i t a t i o n of a m i n o e t h y l g l u t e n i n on S e p h a d e x G - i o o ( c o l u m n 3.8 c m × 155 cm) in o.5 M acetic acid (2 ml of 2 . 5 % solution). H e a v y lines on t h e abscissa indicate c o m b i n e d fractions. B a r g r a p h s below t h e e l u t i o n p a t t e r n s indicate w e i g h t d i s t r i b u t i o n , c a l c u l a t e d on a d r y w e i g h t basis.

Biochim. Biophys. Acta, I6o (1968) 4o4-412

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Chromatography of Fractions A and B from aminoethylglutenin on Sephadex G-Ioo showed that they were mixtures of particles with different molecular weights (Fig. 4). As shown in Fig. 4, components of aminoethylglutenin that migrated with chromatographic mobilities between the two major constituents of crude gliadin (Fig. 3B, Peak II) were concentrated in Fraction A, whereas Fraction B contained high, low, and intermediate molecular weight particles. DISCUSSION

Studies by HABER AND ANFINSEN26 showed that the native configuration of a soluble protein is determined primarily by its amino acid sequence. Nevertheless, it is well known that environmental conditions or chemicals that disrupt the associative interactions of amino acid side chains in proteins can produce substantial changes in the properties of a protein. In cereal grain proteins, which may be subject to extreme temperatures or dehydration during seed maturation, the relative influence of primary structure versus environmental factors is significant in terms of understanding the different physicochemical properties of proteins like glutenin and gliadin. Both our data and compositional differences noted by EWART27between glutenin and gliadin support the contention that glutenin contains some proteins or polypeptides distinctly different from gliadin that may be responsible for its unique properties and disulfide structure. The possibility that the proteins isolated in our Fraction A resulted from selective purification of constituents yet unrecognized in gliadin cannot be excluded, but it seems unlikely in view of the fact that materials in Fraction A had chromatographic properties different from gliadin proteins (Fig. 4). The unusual composition of Fraction A is interesting in terms of the importance of disulfide bonds in glutenin 5. On a molecular basis, gel permeation chromatography suggested that Fraction A proteins have apparent molecular weights near 80 ooo. As such, an average molecule might contain four residues of cysteine, which would be compatible with the concept that glutenin consists of proteins that are disulfide bonded both inter- and intramolecularly ~. If, however, the real molecular weights of fundamental glutenin subunits are near 20 ooo (ref. 5), approx. 28% (Fraction A plus Fraction C) of the molecules might average only one residue of cysteine. Such molecules could occupy terminal positions in a disulfide network in glutenin but could not extend the glutenin matrix unless they were adapted to crosslinking bv associative forces or other forms of bonding.

ACKNOWLEDGEMENT

We are indebted to JAMES CAVINS and CHRISTOPHERJAMES for the amino acid analyses. REFERENCES I M. C. MARKLE¥, Cereal Chem., t 5 (i938) 438. 2 j. s. WALL, in H. W. SCIIULTZ AND A. F. ANGLEMIER, Proteins and Their Reactions, Avi, W e s t p o r t , Conn., 1964, p. 315 . 3 J. H. WOYCHIK AND F. R. HUEBNER, Biochim. Biophys. Acta, 127 (I966) 88. 4 F. R. HUEBNER, J. A. ROTHFUS AND J. S. WALL, Cereal Chem., 44 (1967) 22i.

Biochim. Biophys. Acta, 151 (1968) 404 4r2

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412 5 0 7 8 9 io II I2 13 14 1.5 10 17 18 19 20 2[ 22 23 24 25 26 27

H. C. NIELSFN, G. E. BABCOCK AND F. i{. SENTI, Arch. Biochem. Biophys., 9~) (lq*~2) - 5 2 . J. H. WOYCHIK, I;. R. HUEBNER .and [{. J. |)IMLER, Arch. Biochem. Biophys., lO5 (I904) 151. :\" C. BECK\VITH, J. •. WALL ANI) ]~. W . JORDAN, Arch. Biochem. Biophys., 1 , 2 (10651 ,(~. A. ('. BECKWITH AND J. s. \VALL, Biochirn. Biophys. 4cla, 13o (19661 155. M. A. RAFTERY AND R. D. COLE, J. Biol. Chem., 241 (1966) 3457. R. W . J o n E s , N. W . TAYLOR AND F. R. SENTI, Arch. Biochem. Biophys., 84 (19591 363 . M. J. A. CROW .anD J. A. ROTHFUS, Cereal Chem., in t h e press. l). CAVALLINI, C. DE MARCO, 13. MONDOVI AND (~. F. AZZONE, Experientia, i i (1955) 61. D. H. SPACKMAN, W . H. ~TEIN ANI) ~. MOORE, Anal. Chem., 3 ° (I958) 119o. J. F. CAVINS AND M. FRIEDMAN, Cereal Chem., 45 (1958) 172J. H. WOYCHIK, J. A. ]~OUNI)Y AND R. J. 1)IMLER, Arch. Biochem. Biophys., 94 (196I) 477. A. C. BECKWITH, }~. C. NIELSEN, J. ~. WALL AND t;. R. HUEBNER, CereaIChem., 43 ( t 9 6 6 ) t 4. J. 1~. CARTER, J. Biol. Chem., 234 (19591 17o5. R. E. ]~ENESCH, H . n . LARDY AND R. BENESCH, J. Biol. Chem., 216 (1955) 603J. A. ROTHFUS, Anal. Biochem., 16 ( r 9 6 6 ) I67. S. ROSENBERG, J. C. PERRONE AND P. L. I%IRK, Anal. Chem., 22 (I95O) iiS(). M. SELA, N. L u P u , :\. YARON AND A. BERGER, Biochim. Biophys. Acla, 62 (1962) 594. A. C. ]~ECK~VITH, J. S. WALL AN[) 1~. J. DIMLER, Arch. Biochem. Biophys., I o 3 (x963) 319. J. E. BERNARDIN, D. D. KASARDA AND D. K. MECHAM, J. Biol. Chem., 242 (I967) 445J. H . WOYCHIK, J. A. BOUNDY AND 1~. J. DIMLER, J. Agr. Food Chem., 9 ( I 9 6 I ) 307 • P. ANDREWS, Biochem. J., 9~ (I9641 222. E. HABER AND C. B. ANFINSEN, J. Biol. Chem., 237 (19621 I839. J. A. D. EWART, J. Sei. Food Agr., ~8 (19071 I I 1 .

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