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Reduction and starch-gel electrophoresis of wheat gliadin and glutenin
ARC’HIVES
OF
BIOCHEMISTRY
AXI)
BIOPHYSICS
Reduction
and
Prom
the
ATorthern
(1904)
Starch-Gel
of Wheat J. H. WOTCHIK,
105, 151-155
Gliadin
Electrophoresis and
I’. R. HUEBNER Regional
Received
Glutenin AND
Laboratory,’
Research
September
R. J. DIMLER Peoria,
Illinois
25, 1963
Reduction of the disulfide bonds of wheat gliadin and glutenin followed by electrophoresis in starch gel revealed the presence of some components which are perhaps common to both proteins. There were marked quantitative ditierences in the distribution of components. The release of 20 or more electrophoretic components from the previously unresolved glutenin fraction is further evidence that extensive intermolecular disulfide bonding is responsible for its high molecular weight. Intermolecular disulfide bonding is present only to a limited extent in the gliadin fraction.
tively low moleclular-weight components. Such a mechanism might permit the incorporation of gliadin proteins into the glutenin structure. The present studies were undertaken, therefore, to compare the type of disulfide bonding in gliadin and glutenin, and to determine whether polypeptide or protein chains common to both gluten fractions might be present in the reduced proteins.
The classic wheat gluten fractions, gliadin and glutenin, differ markedly in rheologic properties such as viscosity, elasticity, and cohesiveness (1). These protein fractions differ also in electrophoretic behavior as demonstrated by moving boundary and starch-gel electrophoresis (1, 2). Gliadin, with an average molecular weight of approximately 40,000 (3), is resolved into 7 major components by gel electrophoresis (2). Although glutenin appears homogeneous in moving boundary electrophoresis, it exhibits in the ultracentrifuge a broad spectrum of sedimenting species with an average molecular weight of 223 million (3). Glutenin does not migrate into a starch gel during electrophoresis (a), presumably because of either the high molecular weight or the shape of the protein molecules comprising this fraction. Oxidative cleavage of glutenin yields an ultracentrifugally homogeneous product with a molecular weight of approximately 20,000 (4). This finding suggests that glutenin molecules are formed through extensive intermolecular disulfide bonding of compara-
MATERIALS
AND
METHODS
Gluten was prepared from Ponca hard red winter wheat flour which had been extracted with dry n-butanol at room temperature to remove most of the lipids. The defatted flour was made into dough and washed as described by Jones et al. (1). After inactivation of proteolytic enzymes by heating to 100°C. in 0.01 M acetic acid, the preparation was lyophilized. Gliadin and glutenin were prepared from gluten by fractional alcoholic precipitation of glutenin. A solution of 1 g. of gluten in 50 ml. of 0.01 dl acetic acid was diluted with absolute ethanol to 700,1, ethanol (v/v). The solution was adjusted to a pH meter reading of 0.7 with 1 X sodium hydroxide and placed in a refrigerator for at least 4 hours. The precipitated glutenin was removed by centrifugation, redissolved in 0.01 N acetic acid, and reprecipitated. The supernatant gliadin fraction was dialyzed against, acetic acid, and lyophilized. Examination of the gliadin and glutenin fract,ions by starch-gel
i This is a laboratory of t’he Nort,hern ITtilization Research and Development Division, Agricultural Research Service, 1:. S. Department of Agriculture. 151
152
WOYCHIK,
HUEBNER
electrophoresis (2) showed no cross contamination in the two fractions. The gluten proteins were reduced in 8 M urea and 0.1 M phosphate buffer, pH 8.0, with a 50 M excess of mercaptoethanol based on the disulfide content. Reductions were allowed to proceed for 1 hour before adding the alkylating agent. In the case of iodoacetamide (RCAM-protein), a 25 M excess (based on total SH) of the reagent was added to the reduction mixture, and alkylation was allowed to proceed for 30 minutes. The solution was adjusted to pH 3.5 with acetic acid and the solution dialyzed. A similar procedure was used for alkylation with acrylonitrile (cyanoethylated or RCNE-protein) except that the molar ratio of mercaptoethanol to acrylonitrile was 1:2 (5). Reduction and alkylation were complete in the RCAM-protein; this was indicated by the agreement between previously determined cysteic acid and S-carboxymethylcysteine quantitatively determined in RCAM-hydrolyzates by the procedure of Marko (6). Quantitative determination of S-carboxyethylcysteine in the RCNE-protein by the ion-exchange procedure of Moore et al. (7) has been reported by Weil and Seibles (5). However, in RCNE-gluten hydrolyzates, the S-carboxyethylcysteine peak could not be detected due to the overlap of the very large glutamic acid peak (approximately 40”/ by weight). Amperometric titration (8) with bisulfite, however, indicated the absence of either sulfhydryl or disulfide groups in the alkylated proteins. Starch-gel electrophoresis was performed according to the procedure previously reported (2). Migration was at 7 volts per centimeter (lo15 ma.) for approximately 16 hours. Safranin 0 was added as a reference substance. The reduced state of the nonalkylated proteins was maintained by the inclusion of 0.05 M mercaptoethanol in the starch gel and buffer system. The patterns were detected by staining 1-5 minutes with a 0.1% aqueous solution of nigrosin. As reported by Coulson and Sim (9), nigrosin stains gluten proteins better than amido-black. Individual gliadin components were isolated from lightly stained starch gels. The corresponding bands were cut out and the stained portion of the gel was trimmed away. The protein was removed from the gel by diffusion after maceration in aluminum lactate-3 M urea (2). RESULTS
AND
DISCUSSION
Several methods exist for cleavage of disulfide bonds in proteins (10-13) ; however, not all are readily applicable to proteins on which further structural studies are contemplated. The presence of appreciable
AND
DIMLER
amounts of tryptophan (14) in gluten proteins warrants the exclusion of oxidative techniques; similarly, the alkaline lability of gluten sulfur demands the use of relatively gently reductive techniques. Mercaptoethanol at pH 8.0 in 8 Ad urea was chosen for reduction of gliadin and glutenin. Sulfhydryl groups liberated by reductive techniques are usually alkylated to prevent reoxidation. However, in some instances, alkylation can produce artifacts through reactions with amino groups and methionine sulfur (15). Therefore, electrophoretic patterns of reduced gliadin and glutenin were obtained for comparison with those of their alkylated derivatives. No significant difference in the patterns or in the mobilities of the reduced proteins compared with the respective RCNE-proteins could be seen. In Fig. 1 are presented the electrophoretic patterns of gliadin and glutenin after reduction. A schematic diagram obtained from a compilation of several patterns of the reduced proteins is presented in Fig. 2 to define more clearly the number and relative intensities of the electrophoretic components. Varying the reduction time from 15 minutes to 24 hours did not result in an electrophoretie pattern different from that shown in Figs. 1 and 2. The gliadin fraction showed no apparent increase in the number or change in proportion of major components following reduction. The gliadin components appeared to have been altered structurally, however, as indicated by a reduction of approximately 20 % in their electrophoretic mobilities compared to the native protein. The decreased mobilities, which result from reduction, can probably be attributed to the unfolding of the peptide chain after cleavage of the intramolecular bonds. These unfolded molecules could have more difficutly in passing through the gel pores. Reduction of glutenin, on the other hand, released 20 or more migrating components from a protein that showed no migration in the gel before reduction. Over half of these components are present in only trace amounts and have mobilities greater than that of the fastest gliadin component. The major portion of the glutenin protein can be accounted for by
REDUCTION
OF
WHEAT
GLIADIN
AND
GLUTENIN
Gliadin
Glutenin
FIG. 2. Schematic diagram showing the number and relative intensities of electrophoretic components obtained after reduction of gliadin and glutenin (nonalkylated).
FIG. 1. Starch-gel electrophoretic patterns reduced glisdin and glutenin (nonalkylated). The aluminum lactate buffer system was 0.05 in mercaptoethanol. A, gliadin; B, glutenin.
of &f
the seven principal components shown in Fig. 2. A comparison of electrophoretic patterns, such as those in Figs. 1 and 2, indicates the presence of components in reduced glutenin having counterparts with identical mobilities in reduced gliadin. The exceptions are trace components that have electrophoretic mobjlities greater than the fastest gliadin component. These trace components, not shown in Fig. 2, probably originate from incorporated components comparable to, or identical
154
WOYCHIK,
HUEBNER
with, water-soluble proteins of wheat, which have relatively high electrophoretic mobilities (2). Although major differences in protein distribution are evident among the components of reduced gliadin and reduced glutenin, the finding of components with identical mobilities within these fractions suggests that glutenin arises primarily through intermolecular disulfide bonding of gliadin components. The difference in distribution could result from several causes, such as selectivity in the formation of intermolecular disulfide bonds or the presence of different distribution of gliadin components at the time or site of glutenin synthesis. Water-soluble proteins are probably also incorporated into the glutenin structure, although to a lesser degree. The amino acid compositions and other characteristics of
FIG. 3. Starch-gel
electrophoretic
patterns
AND
DIMLER
components with identical electrophoretic mobilities isolated from reduced gliadin and reduced glutenin will have to be determined to establish whether glutenin contains gliadin components. Further investigations required preparation of the reduced proteins in a form in which the liberated sulfhydryl groups are protected from reoxidation. AcryIonitriIe was selected for this purpose since it did not alter the mobilities of the reduced proteins, in contrast with iodoacetamide, which caused a marked increase in positive charge, presumably because of side reactions with methionine (16). Although the electrophoretic pattern of reduced gliadin (Fig. 1) did not show an apparent increase in number of components, it is possible that the complexity of the frac-
of native
gliadin
and
of reduced
components.
REDUCTION
OF
WHEAT
GLIADIN
tion may have obscured some of the results of disulfide cleavage. Since we were particularly interested in determining whether individual gliadin components consisted of more than one polypeptide chain, these components were isolated from preparative starch gels and reduced. The results obtained after the reduction and alkylation of five gliadin components are presented in Fig. 3, together with a pattern of native gliadin used as a mobility reference. The electrophoretic mobilities of the reduced components have been decreased by 1520% compared with the native proteins. As mentioned previously, the decreased mobilities probably result from configurational changes accompanying reduction. Three of the components, o(,, 01~, and y, continue to migrate as single components after reduction. The p3 and fi4 components (not shown) behaved similarly. These results are indicative of single-chain structures containing only intramolecular disulfide bonds. On the other hand, the p1 and /32 proteins indicated the presence of interchain disulfide bonds by each yielding two corn poncnts with very similar nrobilities after reduction. These studies, therefore, establish that gliadin and glutenin differ primarily in regard to the nature of their disulfide bonding. Intermolecular bonding occurs to a limited extent among the gliadin proteins, whereas it is a principal factor in glutenin structure. The intermolecular bonding of units comparable to gliadin and watersoluble proteins results in a high molecular-
AND
155
GLUTENIN
weight polymer. The origin of some of the unique rheologic properties of glutenin can probably be attributed to the physical state resulting from such polymerization. The gliadin proteins, being much simpler molecules, may function primarily as modifiers of glutenin properties by participating in disulfide interchange reactions. REFERENCES 1. JONES, R. W., TAYLOR, N. W., AND SENTI, F. R., Arch. Biochem. Biophys. 84, 363 (1959). 2. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., Arch. Biochem. Biophys. 94,477 (1961). 3. JONES, R. W., BABCOCK, G. E., TAYLOR, N. W., AND SEKTI, F. It., Arch. Biochem. Biophys. 94, 483 (1961). 4. NIELSEN, H. C., BABCOCK, G. E., AND SENTI, F. R., ,4rch. Biochem. Biophys. 96,252 (1962). 5. WEIL, L., AND SEIBLES, T. S., Arch. B&hem. Biophys. 96, 470 (1961). 6. MARKO, A. M., Can. J. Biochem. Physiol. 36, 1249 (1957). 7. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Snal. Chem. 30, 1185 (1958). 8. CARTER, J. It., J. Biol. Chem. 234, 1705 (1959). 9. CO~JLSON, C. B., AND SIM, A. K., Biochem. J. 80, 1Gp (1961). 10. SANGER, F., Biochem.J.44,126 (1949). 11. HIRS, C. H. W., J. Biol. Chem. 219,611 (1956). 12. SWAN, J. M., Nature 180,643 (1957). 13. STARK, G. R., STEIN, W. H., .&ND MOORE, S., J. Biol. Chem. 236,436 (1961). 14. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., J. Agr. Food Chem. 9,307 (1961). 15. GUNDLACH, H. G., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 234, 1754 (1959). 16.
GUNDLACH,
H., J. Biol.
II.
G.,
Chem.
MOORE,
234,176l
S., AND
(1959).
STEIN,
W.
OF
BIOCHEMISTRY
AXI)
BIOPHYSICS
Reduction
and
Prom
the
ATorthern
(1904)
Starch-Gel
of Wheat J. H. WOTCHIK,
105, 151-155
Gliadin
Electrophoresis and
I’. R. HUEBNER Regional
Received
Glutenin AND
Laboratory,’
Research
September
R. J. DIMLER Peoria,
Illinois
25, 1963
Reduction of the disulfide bonds of wheat gliadin and glutenin followed by electrophoresis in starch gel revealed the presence of some components which are perhaps common to both proteins. There were marked quantitative ditierences in the distribution of components. The release of 20 or more electrophoretic components from the previously unresolved glutenin fraction is further evidence that extensive intermolecular disulfide bonding is responsible for its high molecular weight. Intermolecular disulfide bonding is present only to a limited extent in the gliadin fraction.
tively low moleclular-weight components. Such a mechanism might permit the incorporation of gliadin proteins into the glutenin structure. The present studies were undertaken, therefore, to compare the type of disulfide bonding in gliadin and glutenin, and to determine whether polypeptide or protein chains common to both gluten fractions might be present in the reduced proteins.
The classic wheat gluten fractions, gliadin and glutenin, differ markedly in rheologic properties such as viscosity, elasticity, and cohesiveness (1). These protein fractions differ also in electrophoretic behavior as demonstrated by moving boundary and starch-gel electrophoresis (1, 2). Gliadin, with an average molecular weight of approximately 40,000 (3), is resolved into 7 major components by gel electrophoresis (2). Although glutenin appears homogeneous in moving boundary electrophoresis, it exhibits in the ultracentrifuge a broad spectrum of sedimenting species with an average molecular weight of 223 million (3). Glutenin does not migrate into a starch gel during electrophoresis (a), presumably because of either the high molecular weight or the shape of the protein molecules comprising this fraction. Oxidative cleavage of glutenin yields an ultracentrifugally homogeneous product with a molecular weight of approximately 20,000 (4). This finding suggests that glutenin molecules are formed through extensive intermolecular disulfide bonding of compara-
MATERIALS
AND
METHODS
Gluten was prepared from Ponca hard red winter wheat flour which had been extracted with dry n-butanol at room temperature to remove most of the lipids. The defatted flour was made into dough and washed as described by Jones et al. (1). After inactivation of proteolytic enzymes by heating to 100°C. in 0.01 M acetic acid, the preparation was lyophilized. Gliadin and glutenin were prepared from gluten by fractional alcoholic precipitation of glutenin. A solution of 1 g. of gluten in 50 ml. of 0.01 dl acetic acid was diluted with absolute ethanol to 700,1, ethanol (v/v). The solution was adjusted to a pH meter reading of 0.7 with 1 X sodium hydroxide and placed in a refrigerator for at least 4 hours. The precipitated glutenin was removed by centrifugation, redissolved in 0.01 N acetic acid, and reprecipitated. The supernatant gliadin fraction was dialyzed against, acetic acid, and lyophilized. Examination of the gliadin and glutenin fract,ions by starch-gel
i This is a laboratory of t’he Nort,hern ITtilization Research and Development Division, Agricultural Research Service, 1:. S. Department of Agriculture. 151
152
WOYCHIK,
HUEBNER
electrophoresis (2) showed no cross contamination in the two fractions. The gluten proteins were reduced in 8 M urea and 0.1 M phosphate buffer, pH 8.0, with a 50 M excess of mercaptoethanol based on the disulfide content. Reductions were allowed to proceed for 1 hour before adding the alkylating agent. In the case of iodoacetamide (RCAM-protein), a 25 M excess (based on total SH) of the reagent was added to the reduction mixture, and alkylation was allowed to proceed for 30 minutes. The solution was adjusted to pH 3.5 with acetic acid and the solution dialyzed. A similar procedure was used for alkylation with acrylonitrile (cyanoethylated or RCNE-protein) except that the molar ratio of mercaptoethanol to acrylonitrile was 1:2 (5). Reduction and alkylation were complete in the RCAM-protein; this was indicated by the agreement between previously determined cysteic acid and S-carboxymethylcysteine quantitatively determined in RCAM-hydrolyzates by the procedure of Marko (6). Quantitative determination of S-carboxyethylcysteine in the RCNE-protein by the ion-exchange procedure of Moore et al. (7) has been reported by Weil and Seibles (5). However, in RCNE-gluten hydrolyzates, the S-carboxyethylcysteine peak could not be detected due to the overlap of the very large glutamic acid peak (approximately 40”/ by weight). Amperometric titration (8) with bisulfite, however, indicated the absence of either sulfhydryl or disulfide groups in the alkylated proteins. Starch-gel electrophoresis was performed according to the procedure previously reported (2). Migration was at 7 volts per centimeter (lo15 ma.) for approximately 16 hours. Safranin 0 was added as a reference substance. The reduced state of the nonalkylated proteins was maintained by the inclusion of 0.05 M mercaptoethanol in the starch gel and buffer system. The patterns were detected by staining 1-5 minutes with a 0.1% aqueous solution of nigrosin. As reported by Coulson and Sim (9), nigrosin stains gluten proteins better than amido-black. Individual gliadin components were isolated from lightly stained starch gels. The corresponding bands were cut out and the stained portion of the gel was trimmed away. The protein was removed from the gel by diffusion after maceration in aluminum lactate-3 M urea (2). RESULTS
AND
DISCUSSION
Several methods exist for cleavage of disulfide bonds in proteins (10-13) ; however, not all are readily applicable to proteins on which further structural studies are contemplated. The presence of appreciable
AND
DIMLER
amounts of tryptophan (14) in gluten proteins warrants the exclusion of oxidative techniques; similarly, the alkaline lability of gluten sulfur demands the use of relatively gently reductive techniques. Mercaptoethanol at pH 8.0 in 8 Ad urea was chosen for reduction of gliadin and glutenin. Sulfhydryl groups liberated by reductive techniques are usually alkylated to prevent reoxidation. However, in some instances, alkylation can produce artifacts through reactions with amino groups and methionine sulfur (15). Therefore, electrophoretic patterns of reduced gliadin and glutenin were obtained for comparison with those of their alkylated derivatives. No significant difference in the patterns or in the mobilities of the reduced proteins compared with the respective RCNE-proteins could be seen. In Fig. 1 are presented the electrophoretic patterns of gliadin and glutenin after reduction. A schematic diagram obtained from a compilation of several patterns of the reduced proteins is presented in Fig. 2 to define more clearly the number and relative intensities of the electrophoretic components. Varying the reduction time from 15 minutes to 24 hours did not result in an electrophoretie pattern different from that shown in Figs. 1 and 2. The gliadin fraction showed no apparent increase in the number or change in proportion of major components following reduction. The gliadin components appeared to have been altered structurally, however, as indicated by a reduction of approximately 20 % in their electrophoretic mobilities compared to the native protein. The decreased mobilities, which result from reduction, can probably be attributed to the unfolding of the peptide chain after cleavage of the intramolecular bonds. These unfolded molecules could have more difficutly in passing through the gel pores. Reduction of glutenin, on the other hand, released 20 or more migrating components from a protein that showed no migration in the gel before reduction. Over half of these components are present in only trace amounts and have mobilities greater than that of the fastest gliadin component. The major portion of the glutenin protein can be accounted for by
REDUCTION
OF
WHEAT
GLIADIN
AND
GLUTENIN
Gliadin
Glutenin
FIG. 2. Schematic diagram showing the number and relative intensities of electrophoretic components obtained after reduction of gliadin and glutenin (nonalkylated).
FIG. 1. Starch-gel electrophoretic patterns reduced glisdin and glutenin (nonalkylated). The aluminum lactate buffer system was 0.05 in mercaptoethanol. A, gliadin; B, glutenin.
of &f
the seven principal components shown in Fig. 2. A comparison of electrophoretic patterns, such as those in Figs. 1 and 2, indicates the presence of components in reduced glutenin having counterparts with identical mobilities in reduced gliadin. The exceptions are trace components that have electrophoretic mobjlities greater than the fastest gliadin component. These trace components, not shown in Fig. 2, probably originate from incorporated components comparable to, or identical
154
WOYCHIK,
HUEBNER
with, water-soluble proteins of wheat, which have relatively high electrophoretic mobilities (2). Although major differences in protein distribution are evident among the components of reduced gliadin and reduced glutenin, the finding of components with identical mobilities within these fractions suggests that glutenin arises primarily through intermolecular disulfide bonding of gliadin components. The difference in distribution could result from several causes, such as selectivity in the formation of intermolecular disulfide bonds or the presence of different distribution of gliadin components at the time or site of glutenin synthesis. Water-soluble proteins are probably also incorporated into the glutenin structure, although to a lesser degree. The amino acid compositions and other characteristics of
FIG. 3. Starch-gel
electrophoretic
patterns
AND
DIMLER
components with identical electrophoretic mobilities isolated from reduced gliadin and reduced glutenin will have to be determined to establish whether glutenin contains gliadin components. Further investigations required preparation of the reduced proteins in a form in which the liberated sulfhydryl groups are protected from reoxidation. AcryIonitriIe was selected for this purpose since it did not alter the mobilities of the reduced proteins, in contrast with iodoacetamide, which caused a marked increase in positive charge, presumably because of side reactions with methionine (16). Although the electrophoretic pattern of reduced gliadin (Fig. 1) did not show an apparent increase in number of components, it is possible that the complexity of the frac-
of native
gliadin
and
of reduced
components.
REDUCTION
OF
WHEAT
GLIADIN
tion may have obscured some of the results of disulfide cleavage. Since we were particularly interested in determining whether individual gliadin components consisted of more than one polypeptide chain, these components were isolated from preparative starch gels and reduced. The results obtained after the reduction and alkylation of five gliadin components are presented in Fig. 3, together with a pattern of native gliadin used as a mobility reference. The electrophoretic mobilities of the reduced components have been decreased by 1520% compared with the native proteins. As mentioned previously, the decreased mobilities probably result from configurational changes accompanying reduction. Three of the components, o(,, 01~, and y, continue to migrate as single components after reduction. The p3 and fi4 components (not shown) behaved similarly. These results are indicative of single-chain structures containing only intramolecular disulfide bonds. On the other hand, the p1 and /32 proteins indicated the presence of interchain disulfide bonds by each yielding two corn poncnts with very similar nrobilities after reduction. These studies, therefore, establish that gliadin and glutenin differ primarily in regard to the nature of their disulfide bonding. Intermolecular bonding occurs to a limited extent among the gliadin proteins, whereas it is a principal factor in glutenin structure. The intermolecular bonding of units comparable to gliadin and watersoluble proteins results in a high molecular-
AND
155
GLUTENIN
weight polymer. The origin of some of the unique rheologic properties of glutenin can probably be attributed to the physical state resulting from such polymerization. The gliadin proteins, being much simpler molecules, may function primarily as modifiers of glutenin properties by participating in disulfide interchange reactions. REFERENCES 1. JONES, R. W., TAYLOR, N. W., AND SENTI, F. R., Arch. Biochem. Biophys. 84, 363 (1959). 2. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., Arch. Biochem. Biophys. 94,477 (1961). 3. JONES, R. W., BABCOCK, G. E., TAYLOR, N. W., AND SEKTI, F. It., Arch. Biochem. Biophys. 94, 483 (1961). 4. NIELSEN, H. C., BABCOCK, G. E., AND SENTI, F. R., ,4rch. Biochem. Biophys. 96,252 (1962). 5. WEIL, L., AND SEIBLES, T. S., Arch. B&hem. Biophys. 96, 470 (1961). 6. MARKO, A. M., Can. J. Biochem. Physiol. 36, 1249 (1957). 7. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Snal. Chem. 30, 1185 (1958). 8. CARTER, J. It., J. Biol. Chem. 234, 1705 (1959). 9. CO~JLSON, C. B., AND SIM, A. K., Biochem. J. 80, 1Gp (1961). 10. SANGER, F., Biochem.J.44,126 (1949). 11. HIRS, C. H. W., J. Biol. Chem. 219,611 (1956). 12. SWAN, J. M., Nature 180,643 (1957). 13. STARK, G. R., STEIN, W. H., .&ND MOORE, S., J. Biol. Chem. 236,436 (1961). 14. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., J. Agr. Food Chem. 9,307 (1961). 15. GUNDLACH, H. G., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 234, 1754 (1959). 16.
GUNDLACH,
H., J. Biol.
II.
G.,
Chem.
MOORE,
234,176l
S., AND
(1959).
STEIN,
W.