ANALYTICAL
BIOCHEMISTRY
77, 152- 157 (1977)
N4-Aminoalkyl-Cytidine Ribonuclease
Derivatives: Ligands Affinity Adsorbent9
ROLFE E. SCOFIELD,ROBERT Department
of
P. WERNER, ANDFINN
for WOLD
Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 Received May 17, 1976; accepted September 14, 1976
The bisulfite-induced transamination at the Cposition of cytosine was used to prepare N4-aminoalkyl-cytidine 2’(3’)-monophosphate derivatives in a single, simple reaction. The primary aliphatic amine function of these derivatives could readily be coupled to either cyanogen bromide-activated agarose or a commercially available activated Sepharose derivative (the N-hydroxysuccinimide ester of SepharoseXLaminohexanoic acid) to yield effective and specific affinity adsorbents for bovine pancreatic ribonuclease.
The use of derivatives of nucleosides and nucleotides as specific adsorbents in affinity chromatography (1) has been somewhat limited, presumably because fairly extensive chemical manipulations often are required to prepare the proper derivatives for column attachment (2,3). Thus, in the specific case of preparing affinity adsorbents for pancreatic ribonuclease, attempts to couple cytidine 2’-phosphate directly to cyanogen bromide-activated Sepharose through the 4-amino group of cytosine were unsuccessful, and the preparation of an effective ribonuclease affinity adsorbent required the synthesis of the derivative, 5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate, which could successfully be coupled to Sepharose (2). In this paper we report a simple, one-step reaction by which amino alkyl groups can be incorporated into the 4-position of cytidine derivatives to provide the properly reactive primary amine function required in the coupling to many different activated matrices. The method which is based on the bisulfite-induced transamination at the 4-position (4) is illustrated in Scheme I. It should be of general utility in preparing affinity adsorbents with any cytidine and deoxycytidine derivative. In this work it was used to prepare effective ribonuclease afhnity adsorbents which will be briefly described. EXPERIMENTAL
PROCEDURE
Materials and Assays Bio-Gel A-15m was obtained from Bio-Rad Laboratories, cyanogen bromide from Pierce Chemical Company, and chymotrypsin from r This work was supported by a U.S. Public Health Service Research Grant (GM 15053) from the National Institutes of Health. 152 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
RIBONUCLEASE
AFFINITY
Scheme
ADSORBENTS
153
I
Worthington Biochemical Corporation. Ribonuclease-A from bovine pancreas (lot 92C-0060), cytidine 2’(3’)-monophosphoric acid and cytidine 2’:3’-cyclic phosphate were purchased from Sigma Chemical Company. Sephadex G-10 and activated CH-Sepharose 4B are products of Pharmacia Fine Chemicals Inc. Agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate RNase aflinity gel was purchased from Miles-Yeda, Ltd. The alkyl diamines were all obtained from Aldrich Chemical Company. High voltage paper electrophoresis was performed on Whatman No. 3 paper in an ammonium formate buffer (0.05 M) at pH 2.50 or in a borate buffer (0.01 M) at pH 8.00. Ribonuclease activity was assayed with cytidine 2’:3’-cyclic phosphate according to the procedure of Frensdorff and Sela (5). Methods Preparation of cytidine derivatives. Two hundred and fifty milligrams of cytidine 2’(3’)-monophosphoric acid were added to an aqueous solution containing a 12.5 M excess of NaHSO, and a 20 M excess of the appropriate alkyl diamine (different diamines from propyl to heptyl were used at different times). The reaction mixture (10 ml) was adjusted to pH 7.0 with concentrated HCl and incubated at 37°C for 6 days. The progress of the reaction could be monitored readily by high voltage paper electrophoresis at pH 2.5. The transamination product ran well ahead (toward the cathode) of the starting material and could be distinguished by having both a positive ninhydrin reaction and an ultraviolet absorption. The reaction mixture was applied directly to a Sephadex G-10 column (135 x 3 cm), equilibrated, and eluted with water
154
SCOFIELD,
WERNER AND WOLD
to separate the product from most of the excess diamine and NaHSO,. The pooled front peak containing all the ultraviolet-absorbing material (the desired product, the side product uridylic acid, and unreacted cytidylic acid) was brought to pH 9.0 with concentrated NaOH and applied to a Dowex l-X8 column (2.5 x 15 cm; acetate form). The column was washed with water (500 ml) followed by an 800~ml linear gradient of acetic acid (l- 100 mM). The desired product eluted in the first 260-nmabsorbing peak nearly halfway through the gradient and was collected and lyophilized. The yield of N4-aminoalkyl-cytidine 2’(3’)monophosphate was in the range of 50-60%. The product in each case migrated as a uniform ultraviolet-absorbing and ninhydrin-positive spot on high voltage electrophoresis at both pH 8.0 and pH 2.5. Preparation
of Affinity Adsorbents
A. Coupling to cyanogen bromide-activated agarose. Bio-Gel A-15m was activated according to the procedure developed by March et al. (6). Ten milliliters (settled volume) of activated gel was added to 20 ml of 0.2 M sodium bicarbonate (pH 9.5) containing 50 mg of N4-(7-aminoheptyl)-cytidine 2’(3’)-monophosphate. The slurry was agitated gently at 4°C for 20 hr. After coupling, the gel was washed with 400 ml each of 0.1 M sodium acetate, pH 4.0; 2 M urea; and 0.1 M sodium bicarbonate, pH 9.0. B. Coupling to activated CH-Sepharose 48. This commercially available derivative is prepared by first coupling 6-aminohexanoic acid to cyanogen bromide-activated Sepharose and then activating the carboxyl groups by conversion to the N-hydroxysuccinimide ester. The ester reacts smoothly with free amino groups under mild conditions. The swelled and settled gel (10 ml) was agitated gently with 50 mg of N4(7-aminoheptyl)-cytidine 2’(3’)-phosphate in 20 ml of 0.1 M sodium bicarbonate buffer, pH 8.0, for 4 hr at 4°C. Excess ligand was washed away with 100 ml each of 0.1 M sodium bicarbonate, pH 8.0; 0.05 M formate buffer, pH 4.0; and 0.4 M sodium chloride. Both affinity adsorbents were stored in suspension in 0.4 M sodium chloride at 4°C. RESULTS AND DISCUSSION
Both the binding capacity and binding affinity for ribonuclease appeared to be optimal with the longer carbon chain diamine. Consequently, the coupling of the iV4-aminoheptyl derivative was described for the preparation of the affinity adsorbents, and the description of the characterization of the affinity adsorbents will also be limited to those made with the heptyl derivative. Since an affinity adsorbent for ribonuclease is already available (2), the properties of the new adsorbents were
RIBONUCLEASE
0
0
6
AFFINITY
ADSORBENTS
16
155
24
FIG. 1. Protein binding to Sepharose-N4-(aminohexanoyl-aminoheptyl)-cytidine 2’(3’)phosphate (A, C, and D), and to agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)phosphate (B). The gels were compared in l-ml columns, and about 2 mg of protein was applied in 0.5 ml of buffer. The elution was carried out at room temperature with 0.1 M sodium acetate, pH 5.2, as the buffer. At vertical arrow a, the elution was changed to 0.25 M sodium acetate, pH 5.2; at b to 4 M sodium chloride in 0.25 M sodium acetate, pH 5.2. One-milliliter fractions were collected and the absorbance at 280 nm was recorded. (A, B), Commercial ribonuclease, about 7% pure; (C), performic acid-oxidized ribonuclease; (D), chymotrypsinogen. The crosshatched areas indicate the fractions containing ribonuclease activity.
also compared to a commercial preparation of this adsorbent, agarose5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate. The amount of ligand bound to each of the three adsorbents was determined by analyzing for organic phosphate (7), and the Bio-Gel A-15maminoheptyl-cytidine derivative, the Sepharose-hexanoyl-aminoheptylcytidine derivative, and the commercial derivative were found to contain 2.5, 6.25, and 2.1 pmol of ligand per ml of settled gel, respectively. The specificity and effectiveness of the affinity adsorbents are illustrated in Figs. 1 and 2. Although the ligand content of the three adsorbents differs, the capacity for ribonuclease binding did not appear to be significantly different. Under optimal conditions, all three preparations bound approximately 5 mg of ribonuclease per ml of settled adsorbent. The results presented in the figures were consequently obtained with amounts of enzyme corresponding to less than half the capacity of the columns. As shown in Fig. 1, the properties of the Sepharose-hexanoyl-aminoheptylcytidine column are identical to those of the commercial agaroseuridine column. Under the conditions of the experiment (0.1 M sodium acetate, pH 5.2) the adsorbent is also specific for active ribonuclease, as neither performic acid-oxidized ribonuclease nor chymotrypsinogen is retarded on the column. This is again in agreement with findings obtained with the uridine adsorbent (2). Under the conditions used in the experiments in Fig. 1, ribonuclease did not bind effectively to the Bio-
156
SCOFIELD,
WERNER AND WOLD
0.4
0
0
6
16
24
0 EFFLUENT
6 (ml)
16
24
FIG. 2. Protein binding to Bio-Gel N4-(aminoheptyl)-cytidine 2’(3’)-phosphate (A,C) and to agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate (B,D). In (A) and (B) the 7% pure ribonuclease was fractionated in 0.02 M sodium acetate buffer, pH 5.2, and changed to 0.2 M acetic acid at a. In C and D, chymotrypsinogen was fractionated in 0.02 M Tris*HCl buffer, pH 8.5, and changed to 0.2 M acetic acid at a. Column monitoring was the same as that given in Fig. 1.
Gel-aminohexyl-cytidine column, however, and it was found necessary to lower the salt concentration in order to obtain optimal binding with this adsorbent. Figure 2 shows the results obtained with 0.02 M sodium acetate, pH 5.2, at which optimal binding, identical to that of the commercial uridine adsorbent was observed. Also shown in Fig. 2 is an example of the nonspecific binding which was observed with all of these adsorbents at high pH. At low ionic strength and at pH 8.5, chymotrypsinogen (up to a total of 10 mg) was bound on all three adsorbents. The elution of ribonuclease from the affinity columns was carried out equally well with high sodium chloride concentration (Fig. 1) or with dilute acetic acid (Fig. 2). Since the workup of the purified enzyme is simpler (lyophilization only) with the acetic acid elution, this is the preferred procedure. It is interesting to note that the longer spacer arm of the hexanoic acid coupled to the aminoheptane group provides a ligand more readily available for binding than does the aminoheptane spacer alone. This appears to be a general phenomenon for most affinity adsorbents (1). It is also interesting that the ribonuclease specificity apparently is not affected by the rather bulky substituents in the N4-position of the pyrimidine ring. The substituent in the 5’-position of the uridine derivative (2) could be expected to resemble sufficiently the normal diester linkage of RNA to be inert in the interaction with the enzyme. We originally thought, however, that the integrity of the ring structure might be more critical for specific binding and only proceeded with the affinity adsorbent preparation after showing that the aminoalkyl derivatives of cytidylic acid were as
RIBONUCLEASE
AFFINITY
ADSORBENTS
157
effective as competitive inhibitors of the enzyme as was cytidylic acid itself. From the results obtained, it appears that these readily produced cytidine derivatives are well suited for the production of affinity adsorbents. Because of the simplicity of the preparation of the derivatives from readily available starting materials, the method is well suited for large-scale preparation, and we have successfully used these adsorbents for preparation of substantial quantities of ribonucleases A, B, C, and D from pancreatic juice. REFERENCES 1. Porath, J., and Kristiansen, T. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), 3rd ed., Vol. 1, pp. 95-178, Academic Press, New York. 2. Wilchek, M., and Gorecki, M. (1%9) Eur. J. Biochem. 11, 491-494. 3. Trayer, I., Trayer, H., Small, D., and Bottomley, R. C. (1974) Biochem. J. 139, 609-623. 4. Shapiro, R., and Weisgras, J. M. (1970) Biochem. Biophys. Res. Comm. 40, 839-843. 5. Frensdorff, A., and Sela, M. (1%7) Eur. J. Biochem. 1, 267-280. 6. March, S. C., Pariklu, I., and Cuatrecasas, P. (1974) Anal. Biochem. 60, 149- 152. 7. Bartlett, G. R. (1959)J. Bid. Chem. 234, 466-468.
BIOCHEMISTRY
77, 152- 157 (1977)
N4-Aminoalkyl-Cytidine Ribonuclease
Derivatives: Ligands Affinity Adsorbent9
ROLFE E. SCOFIELD,ROBERT Department
of
P. WERNER, ANDFINN
for WOLD
Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 Received May 17, 1976; accepted September 14, 1976
The bisulfite-induced transamination at the Cposition of cytosine was used to prepare N4-aminoalkyl-cytidine 2’(3’)-monophosphate derivatives in a single, simple reaction. The primary aliphatic amine function of these derivatives could readily be coupled to either cyanogen bromide-activated agarose or a commercially available activated Sepharose derivative (the N-hydroxysuccinimide ester of SepharoseXLaminohexanoic acid) to yield effective and specific affinity adsorbents for bovine pancreatic ribonuclease.
The use of derivatives of nucleosides and nucleotides as specific adsorbents in affinity chromatography (1) has been somewhat limited, presumably because fairly extensive chemical manipulations often are required to prepare the proper derivatives for column attachment (2,3). Thus, in the specific case of preparing affinity adsorbents for pancreatic ribonuclease, attempts to couple cytidine 2’-phosphate directly to cyanogen bromide-activated Sepharose through the 4-amino group of cytosine were unsuccessful, and the preparation of an effective ribonuclease affinity adsorbent required the synthesis of the derivative, 5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate, which could successfully be coupled to Sepharose (2). In this paper we report a simple, one-step reaction by which amino alkyl groups can be incorporated into the 4-position of cytidine derivatives to provide the properly reactive primary amine function required in the coupling to many different activated matrices. The method which is based on the bisulfite-induced transamination at the 4-position (4) is illustrated in Scheme I. It should be of general utility in preparing affinity adsorbents with any cytidine and deoxycytidine derivative. In this work it was used to prepare effective ribonuclease afhnity adsorbents which will be briefly described. EXPERIMENTAL
PROCEDURE
Materials and Assays Bio-Gel A-15m was obtained from Bio-Rad Laboratories, cyanogen bromide from Pierce Chemical Company, and chymotrypsin from r This work was supported by a U.S. Public Health Service Research Grant (GM 15053) from the National Institutes of Health. 152 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
RIBONUCLEASE
AFFINITY
Scheme
ADSORBENTS
153
I
Worthington Biochemical Corporation. Ribonuclease-A from bovine pancreas (lot 92C-0060), cytidine 2’(3’)-monophosphoric acid and cytidine 2’:3’-cyclic phosphate were purchased from Sigma Chemical Company. Sephadex G-10 and activated CH-Sepharose 4B are products of Pharmacia Fine Chemicals Inc. Agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate RNase aflinity gel was purchased from Miles-Yeda, Ltd. The alkyl diamines were all obtained from Aldrich Chemical Company. High voltage paper electrophoresis was performed on Whatman No. 3 paper in an ammonium formate buffer (0.05 M) at pH 2.50 or in a borate buffer (0.01 M) at pH 8.00. Ribonuclease activity was assayed with cytidine 2’:3’-cyclic phosphate according to the procedure of Frensdorff and Sela (5). Methods Preparation of cytidine derivatives. Two hundred and fifty milligrams of cytidine 2’(3’)-monophosphoric acid were added to an aqueous solution containing a 12.5 M excess of NaHSO, and a 20 M excess of the appropriate alkyl diamine (different diamines from propyl to heptyl were used at different times). The reaction mixture (10 ml) was adjusted to pH 7.0 with concentrated HCl and incubated at 37°C for 6 days. The progress of the reaction could be monitored readily by high voltage paper electrophoresis at pH 2.5. The transamination product ran well ahead (toward the cathode) of the starting material and could be distinguished by having both a positive ninhydrin reaction and an ultraviolet absorption. The reaction mixture was applied directly to a Sephadex G-10 column (135 x 3 cm), equilibrated, and eluted with water
154
SCOFIELD,
WERNER AND WOLD
to separate the product from most of the excess diamine and NaHSO,. The pooled front peak containing all the ultraviolet-absorbing material (the desired product, the side product uridylic acid, and unreacted cytidylic acid) was brought to pH 9.0 with concentrated NaOH and applied to a Dowex l-X8 column (2.5 x 15 cm; acetate form). The column was washed with water (500 ml) followed by an 800~ml linear gradient of acetic acid (l- 100 mM). The desired product eluted in the first 260-nmabsorbing peak nearly halfway through the gradient and was collected and lyophilized. The yield of N4-aminoalkyl-cytidine 2’(3’)monophosphate was in the range of 50-60%. The product in each case migrated as a uniform ultraviolet-absorbing and ninhydrin-positive spot on high voltage electrophoresis at both pH 8.0 and pH 2.5. Preparation
of Affinity Adsorbents
A. Coupling to cyanogen bromide-activated agarose. Bio-Gel A-15m was activated according to the procedure developed by March et al. (6). Ten milliliters (settled volume) of activated gel was added to 20 ml of 0.2 M sodium bicarbonate (pH 9.5) containing 50 mg of N4-(7-aminoheptyl)-cytidine 2’(3’)-monophosphate. The slurry was agitated gently at 4°C for 20 hr. After coupling, the gel was washed with 400 ml each of 0.1 M sodium acetate, pH 4.0; 2 M urea; and 0.1 M sodium bicarbonate, pH 9.0. B. Coupling to activated CH-Sepharose 48. This commercially available derivative is prepared by first coupling 6-aminohexanoic acid to cyanogen bromide-activated Sepharose and then activating the carboxyl groups by conversion to the N-hydroxysuccinimide ester. The ester reacts smoothly with free amino groups under mild conditions. The swelled and settled gel (10 ml) was agitated gently with 50 mg of N4(7-aminoheptyl)-cytidine 2’(3’)-phosphate in 20 ml of 0.1 M sodium bicarbonate buffer, pH 8.0, for 4 hr at 4°C. Excess ligand was washed away with 100 ml each of 0.1 M sodium bicarbonate, pH 8.0; 0.05 M formate buffer, pH 4.0; and 0.4 M sodium chloride. Both affinity adsorbents were stored in suspension in 0.4 M sodium chloride at 4°C. RESULTS AND DISCUSSION
Both the binding capacity and binding affinity for ribonuclease appeared to be optimal with the longer carbon chain diamine. Consequently, the coupling of the iV4-aminoheptyl derivative was described for the preparation of the affinity adsorbents, and the description of the characterization of the affinity adsorbents will also be limited to those made with the heptyl derivative. Since an affinity adsorbent for ribonuclease is already available (2), the properties of the new adsorbents were
RIBONUCLEASE
0
0
6
AFFINITY
ADSORBENTS
16
155
24
FIG. 1. Protein binding to Sepharose-N4-(aminohexanoyl-aminoheptyl)-cytidine 2’(3’)phosphate (A, C, and D), and to agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)phosphate (B). The gels were compared in l-ml columns, and about 2 mg of protein was applied in 0.5 ml of buffer. The elution was carried out at room temperature with 0.1 M sodium acetate, pH 5.2, as the buffer. At vertical arrow a, the elution was changed to 0.25 M sodium acetate, pH 5.2; at b to 4 M sodium chloride in 0.25 M sodium acetate, pH 5.2. One-milliliter fractions were collected and the absorbance at 280 nm was recorded. (A, B), Commercial ribonuclease, about 7% pure; (C), performic acid-oxidized ribonuclease; (D), chymotrypsinogen. The crosshatched areas indicate the fractions containing ribonuclease activity.
also compared to a commercial preparation of this adsorbent, agarose5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate. The amount of ligand bound to each of the three adsorbents was determined by analyzing for organic phosphate (7), and the Bio-Gel A-15maminoheptyl-cytidine derivative, the Sepharose-hexanoyl-aminoheptylcytidine derivative, and the commercial derivative were found to contain 2.5, 6.25, and 2.1 pmol of ligand per ml of settled gel, respectively. The specificity and effectiveness of the affinity adsorbents are illustrated in Figs. 1 and 2. Although the ligand content of the three adsorbents differs, the capacity for ribonuclease binding did not appear to be significantly different. Under optimal conditions, all three preparations bound approximately 5 mg of ribonuclease per ml of settled adsorbent. The results presented in the figures were consequently obtained with amounts of enzyme corresponding to less than half the capacity of the columns. As shown in Fig. 1, the properties of the Sepharose-hexanoyl-aminoheptylcytidine column are identical to those of the commercial agaroseuridine column. Under the conditions of the experiment (0.1 M sodium acetate, pH 5.2) the adsorbent is also specific for active ribonuclease, as neither performic acid-oxidized ribonuclease nor chymotrypsinogen is retarded on the column. This is again in agreement with findings obtained with the uridine adsorbent (2). Under the conditions used in the experiments in Fig. 1, ribonuclease did not bind effectively to the Bio-
156
SCOFIELD,
WERNER AND WOLD
0.4
0
0
6
16
24
0 EFFLUENT
6 (ml)
16
24
FIG. 2. Protein binding to Bio-Gel N4-(aminoheptyl)-cytidine 2’(3’)-phosphate (A,C) and to agarose-5’-(4-aminophenyl-phosphoryl)-uridine 2’(3’)-phosphate (B,D). In (A) and (B) the 7% pure ribonuclease was fractionated in 0.02 M sodium acetate buffer, pH 5.2, and changed to 0.2 M acetic acid at a. In C and D, chymotrypsinogen was fractionated in 0.02 M Tris*HCl buffer, pH 8.5, and changed to 0.2 M acetic acid at a. Column monitoring was the same as that given in Fig. 1.
Gel-aminohexyl-cytidine column, however, and it was found necessary to lower the salt concentration in order to obtain optimal binding with this adsorbent. Figure 2 shows the results obtained with 0.02 M sodium acetate, pH 5.2, at which optimal binding, identical to that of the commercial uridine adsorbent was observed. Also shown in Fig. 2 is an example of the nonspecific binding which was observed with all of these adsorbents at high pH. At low ionic strength and at pH 8.5, chymotrypsinogen (up to a total of 10 mg) was bound on all three adsorbents. The elution of ribonuclease from the affinity columns was carried out equally well with high sodium chloride concentration (Fig. 1) or with dilute acetic acid (Fig. 2). Since the workup of the purified enzyme is simpler (lyophilization only) with the acetic acid elution, this is the preferred procedure. It is interesting to note that the longer spacer arm of the hexanoic acid coupled to the aminoheptane group provides a ligand more readily available for binding than does the aminoheptane spacer alone. This appears to be a general phenomenon for most affinity adsorbents (1). It is also interesting that the ribonuclease specificity apparently is not affected by the rather bulky substituents in the N4-position of the pyrimidine ring. The substituent in the 5’-position of the uridine derivative (2) could be expected to resemble sufficiently the normal diester linkage of RNA to be inert in the interaction with the enzyme. We originally thought, however, that the integrity of the ring structure might be more critical for specific binding and only proceeded with the affinity adsorbent preparation after showing that the aminoalkyl derivatives of cytidylic acid were as
RIBONUCLEASE
AFFINITY
ADSORBENTS
157
effective as competitive inhibitors of the enzyme as was cytidylic acid itself. From the results obtained, it appears that these readily produced cytidine derivatives are well suited for the production of affinity adsorbents. Because of the simplicity of the preparation of the derivatives from readily available starting materials, the method is well suited for large-scale preparation, and we have successfully used these adsorbents for preparation of substantial quantities of ribonucleases A, B, C, and D from pancreatic juice. REFERENCES 1. Porath, J., and Kristiansen, T. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), 3rd ed., Vol. 1, pp. 95-178, Academic Press, New York. 2. Wilchek, M., and Gorecki, M. (1%9) Eur. J. Biochem. 11, 491-494. 3. Trayer, I., Trayer, H., Small, D., and Bottomley, R. C. (1974) Biochem. J. 139, 609-623. 4. Shapiro, R., and Weisgras, J. M. (1970) Biochem. Biophys. Res. Comm. 40, 839-843. 5. Frensdorff, A., and Sela, M. (1%7) Eur. J. Biochem. 1, 267-280. 6. March, S. C., Pariklu, I., and Cuatrecasas, P. (1974) Anal. Biochem. 60, 149- 152. 7. Bartlett, G. R. (1959)J. Bid. Chem. 234, 466-468.
