ANALYTICAL
BIOCHEMISTRY
73, 134-138
(1976)
Chromatography of Renin Substrate on Concanavalin A-Agarose MORTON P. PRINTZ, TIMOTHY J. GREGORY, JOHN A. LEWICKI, AND JANA M. PRINTZ Division
of Pharmacology, Department of Medicine, Room 2042, Basic University of California at San Diego, La Jolla, California,
Science 92093
Building,
Received September 2, 1975; accepted January 26, 1976 Renin substrate, the serum prohormone of angiotensin, is a glycoprotein which is shown to bind to concanavalin A. This interaction permits the use of lectin affinity chromatography in overcoming a major problem in the protein’s purification, namely the separation of renin substrate from albumin and similar serum proteins. Low concentrations of either a-methyl mannoside or o-glucose displace renin substrate from the affinity column with no significant loss of pharmacological activity.
Renin substrate, the prohormone of angiotensin II, is an o-globulin occurring in normal human and animal sera in concentrations of lo-30 pg/ml (1). The protein is the substrate for renin which cleaves the leu,,,-leu,, peptide bond to release the decapeptide angiotensin I from the N-terminus. A subsequent enzymatic cleavage of angiotensin I by converting enzyme (2) generates the pharmacologically active octapeptide, angiotensin II. Little is known about renin substrate primarily due to difficulties in its isolation and purification. A partial characterization of hog renin substrate (3) identified it as a glycoprotein with an apparent molecular weight of 55,000, an isoelectric point around 5.0-5.5 and containing 7- 12 mol of neutral hexoses. In subsequent purification attempts, difficulties have been encountered in separating renin substrate from albumin and other serum proteins. We demonstrate in this paper that this difficulty can be resolved by the use of lectin affinity chromatography. The protein concanavalin A exhibits a high binding affinity for terminal mannose and glucose residues of glycoproteins (4). Thus, when covalently bound to a solid support such as agarose or Sepharose it can selectively adsorb those glycoproteins containing the proper configuration of the carbohydrate ligand (5). Displacement of the bound glycoprotein can be achieved by eluting with various concentrations of a-methyl mannoside, a-methyl glucoside, or D-glucose. MATERIALS
AND METHODS
Renin substrate was partially purified from serum isolated from 48 hr postbilaterally nephrectomized rabbits by cardiac puncture. The blood 134 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHY
OF RENIN
SUBSTRATE
135
was drawn into heparinized syringes containing 0.1 M EDTA. The plasma was obtained by low speed centrifugation and found to contain 0.01% by weight active renin substrate. Partial purification involved differential ammonium sulfate precipitation which included a pH 2.5 acid treatment to inactivate proteases, and gel chromatography using Sephadex G-150, the details to be described in detail elsewhere. The preparation resulting from these three steps contained over 2% by weight active substrate. Renin substrate concentrations were determined by bioassay of the angiotensin 1 released by homologous rabbit renin using the standard pentolinium (Wyeth)-blocked rat pressor preparation (6). Renin was isolated from rabbit kidneys using the procedures of Ryan and MacKenzie (7). In the determination of renin substrate concentrations sufficient renin was used to release all the substrate activity in 3 to 5 hr of incubation at 37°C. The reaction mixture contained 2 mM EDTA, 1 mM phenylmercuric acetate, and 40 mM sodium phosphate buffer, pH 6.0. The data are expressed in terms of angiotensin II equivalents of activity. Concanavalin A-Sepharose (Con A-Sepharose), used for the lectin affinity chromatography, was obtained from Pharmacia Fine Chemicals. As reported by the manufacturer, 10 mg of concanavalin A was covalently bound to each gram of Sepharose 4B (Pharmacia). The buffer contained 10 mM sodium chloride, 10 mM sodium acetate, 1 mM MnCl, and 1 mM CaCl,, pH 6.0. The gel was washed with over 10 volumes of buffer prior to application of the sample. Renin substrate samples always contained EDTA to inhibit divalent cation-activated proteases. Since EDTA was found to interfere with complete binding of the protein to the concanavalin A a IO-fold concentrated aliquot of the acetatecalcium-manganese buffer was added to the sample just prior to its application to the column. Eluates were collected in test tubes containing l/l0 volume of 0.1 M EDTA, pH 6.0, and elution profiles were monitored both by optical density at 280 nm and by bioassay. The protein was loaded onto the column and eluted at 5°C. The quantities of glycoproteins loaded onto any column never exceeded the capacity of the gel to adsorb all the renin substrate in the sample. In independent studies we find that all the substrate in 1.5 to 2.0 mls of plasma are bound by a 1.0 ml Con A-Sepharose column. RESULTS
A Con A-Sepharose column binds 85-95% of the pressor activity of a partially purified rabbit renin substrate preparation as shown in Fig. 1. The first optical density peak contains between 55-70% of the total protein loaded onto the column, as determined by optical density at 280 nm, and represents nonadsorbing proteins. Less than 15% of the angiotensin activity is in this peak. Renin substrate is eluted by com-
136
PRINT2
ET AL.
paratively low concentrations of either a-methyl mannoside or D-glucose. With a stepwise increase of o-methyl mannoside concentration (Fig. 1) 70% of the active renin substrate elutes at 0.001 M while all the activity is eluted by 0.002 M. The displacement of renin substrate is not significantly altered by elution at 25°C or by 1 M NaCl. The results in Fig. 2a indicate that over 85% of the renin substrate activity is eluted between 0.002 and 0.008 M D-glucose. This concentration is approximately 3- to IO-fold greater than that required of c-w-methyl mannoside. The final preparation contains approximately 6- 12% active renin substrate by weight. A minimum 4- to 6-fold purification can be obtained in one chromatographic step. The purification of the active prohormone in the experiment described by Fig. 2a was monitored by polyacrylamide gel electrophoresis using the discontinuous disc method (8,9); results are shown in Fig. 2b. The nonabsorbed protein peak, gel A, contains many bands. Gel B contains protein(s) which are inactive and elute at a concentration of 0.001 M D-agarose. Gel C is the active protein preparation while gel D includes inactive proteins which elute at concentrations of D-glucose greater than 0.008 M. DISCUSSION
The use of lectin affinity chromatography permits a new approach in the partial purification of renin substrate, since it resolves and separates
FIG. 1. Stepwise elution of rabbit renin substrate by o-methyl mannoside. Partially purified renin substrate (6 ml) was loaded onto a 10-m] column. The flow rate was 0.25-0.3 mhmin. Stepwise elution involved addition of 20 ml buffer with varying concentrations of o-methyl mannoside. Optical density was determined on a Beckman Acta spectrophotometer and activity is expressed in terms of angiotensin II equivalents/ml.
CHROMATOGRAPHY
FRACTION
OF RENIN
137
SUBSTRATE
NUMBER
A
6
C
D
FIG. 2. (a) Stepwise elution of renin substrate by o-glucose. Partially purified renin substrate (3 ml) was loaded onto a lO-ml Con A-Sepharose column and eluted as described in Fig. 1. The eluted proteins were pooled according to activity in the following manner: Fraction l-tubes 5 through 20; Fraction 2-tubes 30 through 59; Fraction 3-tubes 60 through 120; Fraction 4-tubes 120 through 180. The various fractions were concentrated by ultrafiltration. (b) Polyacrylamide gel electrophoresis of fractions from the o-glucose elution of renin substrate. Discontinuous polyacrylamide gel electrophoresis at pH 4.5 followed the methods described by Gabriel (6). The separating gel was prerun for 2 hr at 7°C to remove excess ammonium persulfate. The samples were layered in sucrose and pyronin yellow was used as the maker dye. Each concentrated fraction (50 ~1) was layered and electrophoresis conducted at 7°C. The gels were fixed in 12.5% TCA, stained overnight at 5°C in 1% Coomassie blue, and destained in 7% acetic acid. Pattern A is Fraction 1 from Fig. 2a, B-Fraction 2. C-Fraction 3 and D-Fraction 4. Fraction 3 contains 85% of the angiotensin activity.
many proteins with molecular weights and eiectrophoretic mobilities similar to the prohormone. Renin substrate binds to Con A-Sepharose and is selectively eluted by glycosides which exhibit high affinity for concanavalin A. Thus, concanavalin A appears to separate renin substrate by the principles of affinity chromatography. Our data would indicate that among the carbohydrate components present in renin substrate are residues with high affinity for concanavalin A. There is no significant contribution from electrostatic or hydrophobic interactions to the stabilization of a renin substrate-concanavalin A complex, since elevated ionic strength or temperature had little effect on the separation. The displacement of renin substrate by low concentrations of a-methyl would indicate that the specific carbohydrate mannoside or D-ghCOSe interaction is labile. A preparation of partially purified renin substrate prior to concanavalin A chromatography contains many proteins. The majority of these proteins are not absorbed to concanavalin A-Sepharose and elute
138
PRINTZ
ET AL.
immediately from the column. Many of these proteins are electrophoretically indistinguishable from renin substrate which is one reason why prior attempts to purify renin substrate by electrophoretic procedures were only partly successful. The concanavalin A-Sepharose column also separates many glycoproteins from renin substrate. Those proteins which elute after renin substrate are quite different electrophoretically and apparently represent unrelated classes of protein. We find that this method of affinity chromatography is suitable both for partially purified preparations of renin substrate as well as dialyzed serum. ACKNOWLEDGMENT This investigation was supported by Research Grant HL 15808 from the National Institutes of Health and by a grant from the Population Council, New York, Grant No. M74.34.
REFERENCES 1. Page, 1. H., and McCubbin, J. W. (1968) in “Renal Hypertension,” pp. 20-27, Year Book Med. Pub]., Chicago. 2. Yang, H. Y. T., Erdos, E. G., and Levin, Y. (1971) J. Phar. &per. Ther. 177, 291-300. 3. Skeggs, L. T. Jr., Lentz, K. E., Hochstrasser, H., and Kahn, J. R. (1%3) J. Exp. Med. 118, 73-98. 4. Goldstein, I. J., Hollerman, C. E., and Smith, E. E. (1965) Biochemisfry 4, 876883.
5. Cuatrecasas, P., Wilchek, M., and Antinsen, C. B. (1968) Proc. Nut. Acnd. Sci. USA 61, 636-643. 6. Regoli, D., Park, W. K., and Rioux, F. (1974) Pharmncol. Rev. 26, 69-124. 7. Ryan, J. W., and McKenzie, J. K. (1968) Biochem. J. 108, 687-692. 8. Davis, B. J. (1964)Ann. N.Y. Acad. Sci. 121, 404-427. 9. Gabriel, 0. (1971) in “Methods in Enzymology” (Jakoby, W. B., ed.), Vol 12, pp. 565-578, Academic Press, New York.
BIOCHEMISTRY
73, 134-138
(1976)
Chromatography of Renin Substrate on Concanavalin A-Agarose MORTON P. PRINTZ, TIMOTHY J. GREGORY, JOHN A. LEWICKI, AND JANA M. PRINTZ Division
of Pharmacology, Department of Medicine, Room 2042, Basic University of California at San Diego, La Jolla, California,
Science 92093
Building,
Received September 2, 1975; accepted January 26, 1976 Renin substrate, the serum prohormone of angiotensin, is a glycoprotein which is shown to bind to concanavalin A. This interaction permits the use of lectin affinity chromatography in overcoming a major problem in the protein’s purification, namely the separation of renin substrate from albumin and similar serum proteins. Low concentrations of either a-methyl mannoside or o-glucose displace renin substrate from the affinity column with no significant loss of pharmacological activity.
Renin substrate, the prohormone of angiotensin II, is an o-globulin occurring in normal human and animal sera in concentrations of lo-30 pg/ml (1). The protein is the substrate for renin which cleaves the leu,,,-leu,, peptide bond to release the decapeptide angiotensin I from the N-terminus. A subsequent enzymatic cleavage of angiotensin I by converting enzyme (2) generates the pharmacologically active octapeptide, angiotensin II. Little is known about renin substrate primarily due to difficulties in its isolation and purification. A partial characterization of hog renin substrate (3) identified it as a glycoprotein with an apparent molecular weight of 55,000, an isoelectric point around 5.0-5.5 and containing 7- 12 mol of neutral hexoses. In subsequent purification attempts, difficulties have been encountered in separating renin substrate from albumin and other serum proteins. We demonstrate in this paper that this difficulty can be resolved by the use of lectin affinity chromatography. The protein concanavalin A exhibits a high binding affinity for terminal mannose and glucose residues of glycoproteins (4). Thus, when covalently bound to a solid support such as agarose or Sepharose it can selectively adsorb those glycoproteins containing the proper configuration of the carbohydrate ligand (5). Displacement of the bound glycoprotein can be achieved by eluting with various concentrations of a-methyl mannoside, a-methyl glucoside, or D-glucose. MATERIALS
AND METHODS
Renin substrate was partially purified from serum isolated from 48 hr postbilaterally nephrectomized rabbits by cardiac puncture. The blood 134 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHY
OF RENIN
SUBSTRATE
135
was drawn into heparinized syringes containing 0.1 M EDTA. The plasma was obtained by low speed centrifugation and found to contain 0.01% by weight active renin substrate. Partial purification involved differential ammonium sulfate precipitation which included a pH 2.5 acid treatment to inactivate proteases, and gel chromatography using Sephadex G-150, the details to be described in detail elsewhere. The preparation resulting from these three steps contained over 2% by weight active substrate. Renin substrate concentrations were determined by bioassay of the angiotensin 1 released by homologous rabbit renin using the standard pentolinium (Wyeth)-blocked rat pressor preparation (6). Renin was isolated from rabbit kidneys using the procedures of Ryan and MacKenzie (7). In the determination of renin substrate concentrations sufficient renin was used to release all the substrate activity in 3 to 5 hr of incubation at 37°C. The reaction mixture contained 2 mM EDTA, 1 mM phenylmercuric acetate, and 40 mM sodium phosphate buffer, pH 6.0. The data are expressed in terms of angiotensin II equivalents of activity. Concanavalin A-Sepharose (Con A-Sepharose), used for the lectin affinity chromatography, was obtained from Pharmacia Fine Chemicals. As reported by the manufacturer, 10 mg of concanavalin A was covalently bound to each gram of Sepharose 4B (Pharmacia). The buffer contained 10 mM sodium chloride, 10 mM sodium acetate, 1 mM MnCl, and 1 mM CaCl,, pH 6.0. The gel was washed with over 10 volumes of buffer prior to application of the sample. Renin substrate samples always contained EDTA to inhibit divalent cation-activated proteases. Since EDTA was found to interfere with complete binding of the protein to the concanavalin A a IO-fold concentrated aliquot of the acetatecalcium-manganese buffer was added to the sample just prior to its application to the column. Eluates were collected in test tubes containing l/l0 volume of 0.1 M EDTA, pH 6.0, and elution profiles were monitored both by optical density at 280 nm and by bioassay. The protein was loaded onto the column and eluted at 5°C. The quantities of glycoproteins loaded onto any column never exceeded the capacity of the gel to adsorb all the renin substrate in the sample. In independent studies we find that all the substrate in 1.5 to 2.0 mls of plasma are bound by a 1.0 ml Con A-Sepharose column. RESULTS
A Con A-Sepharose column binds 85-95% of the pressor activity of a partially purified rabbit renin substrate preparation as shown in Fig. 1. The first optical density peak contains between 55-70% of the total protein loaded onto the column, as determined by optical density at 280 nm, and represents nonadsorbing proteins. Less than 15% of the angiotensin activity is in this peak. Renin substrate is eluted by com-
136
PRINT2
ET AL.
paratively low concentrations of either a-methyl mannoside or D-glucose. With a stepwise increase of o-methyl mannoside concentration (Fig. 1) 70% of the active renin substrate elutes at 0.001 M while all the activity is eluted by 0.002 M. The displacement of renin substrate is not significantly altered by elution at 25°C or by 1 M NaCl. The results in Fig. 2a indicate that over 85% of the renin substrate activity is eluted between 0.002 and 0.008 M D-glucose. This concentration is approximately 3- to IO-fold greater than that required of c-w-methyl mannoside. The final preparation contains approximately 6- 12% active renin substrate by weight. A minimum 4- to 6-fold purification can be obtained in one chromatographic step. The purification of the active prohormone in the experiment described by Fig. 2a was monitored by polyacrylamide gel electrophoresis using the discontinuous disc method (8,9); results are shown in Fig. 2b. The nonabsorbed protein peak, gel A, contains many bands. Gel B contains protein(s) which are inactive and elute at a concentration of 0.001 M D-agarose. Gel C is the active protein preparation while gel D includes inactive proteins which elute at concentrations of D-glucose greater than 0.008 M. DISCUSSION
The use of lectin affinity chromatography permits a new approach in the partial purification of renin substrate, since it resolves and separates
FIG. 1. Stepwise elution of rabbit renin substrate by o-methyl mannoside. Partially purified renin substrate (6 ml) was loaded onto a 10-m] column. The flow rate was 0.25-0.3 mhmin. Stepwise elution involved addition of 20 ml buffer with varying concentrations of o-methyl mannoside. Optical density was determined on a Beckman Acta spectrophotometer and activity is expressed in terms of angiotensin II equivalents/ml.
CHROMATOGRAPHY
FRACTION
OF RENIN
137
SUBSTRATE
NUMBER
A
6
C
D
FIG. 2. (a) Stepwise elution of renin substrate by o-glucose. Partially purified renin substrate (3 ml) was loaded onto a lO-ml Con A-Sepharose column and eluted as described in Fig. 1. The eluted proteins were pooled according to activity in the following manner: Fraction l-tubes 5 through 20; Fraction 2-tubes 30 through 59; Fraction 3-tubes 60 through 120; Fraction 4-tubes 120 through 180. The various fractions were concentrated by ultrafiltration. (b) Polyacrylamide gel electrophoresis of fractions from the o-glucose elution of renin substrate. Discontinuous polyacrylamide gel electrophoresis at pH 4.5 followed the methods described by Gabriel (6). The separating gel was prerun for 2 hr at 7°C to remove excess ammonium persulfate. The samples were layered in sucrose and pyronin yellow was used as the maker dye. Each concentrated fraction (50 ~1) was layered and electrophoresis conducted at 7°C. The gels were fixed in 12.5% TCA, stained overnight at 5°C in 1% Coomassie blue, and destained in 7% acetic acid. Pattern A is Fraction 1 from Fig. 2a, B-Fraction 2. C-Fraction 3 and D-Fraction 4. Fraction 3 contains 85% of the angiotensin activity.
many proteins with molecular weights and eiectrophoretic mobilities similar to the prohormone. Renin substrate binds to Con A-Sepharose and is selectively eluted by glycosides which exhibit high affinity for concanavalin A. Thus, concanavalin A appears to separate renin substrate by the principles of affinity chromatography. Our data would indicate that among the carbohydrate components present in renin substrate are residues with high affinity for concanavalin A. There is no significant contribution from electrostatic or hydrophobic interactions to the stabilization of a renin substrate-concanavalin A complex, since elevated ionic strength or temperature had little effect on the separation. The displacement of renin substrate by low concentrations of a-methyl would indicate that the specific carbohydrate mannoside or D-ghCOSe interaction is labile. A preparation of partially purified renin substrate prior to concanavalin A chromatography contains many proteins. The majority of these proteins are not absorbed to concanavalin A-Sepharose and elute
138
PRINTZ
ET AL.
immediately from the column. Many of these proteins are electrophoretically indistinguishable from renin substrate which is one reason why prior attempts to purify renin substrate by electrophoretic procedures were only partly successful. The concanavalin A-Sepharose column also separates many glycoproteins from renin substrate. Those proteins which elute after renin substrate are quite different electrophoretically and apparently represent unrelated classes of protein. We find that this method of affinity chromatography is suitable both for partially purified preparations of renin substrate as well as dialyzed serum. ACKNOWLEDGMENT This investigation was supported by Research Grant HL 15808 from the National Institutes of Health and by a grant from the Population Council, New York, Grant No. M74.34.
REFERENCES 1. Page, 1. H., and McCubbin, J. W. (1968) in “Renal Hypertension,” pp. 20-27, Year Book Med. Pub]., Chicago. 2. Yang, H. Y. T., Erdos, E. G., and Levin, Y. (1971) J. Phar. &per. Ther. 177, 291-300. 3. Skeggs, L. T. Jr., Lentz, K. E., Hochstrasser, H., and Kahn, J. R. (1%3) J. Exp. Med. 118, 73-98. 4. Goldstein, I. J., Hollerman, C. E., and Smith, E. E. (1965) Biochemisfry 4, 876883.
5. Cuatrecasas, P., Wilchek, M., and Antinsen, C. B. (1968) Proc. Nut. Acnd. Sci. USA 61, 636-643. 6. Regoli, D., Park, W. K., and Rioux, F. (1974) Pharmncol. Rev. 26, 69-124. 7. Ryan, J. W., and McKenzie, J. K. (1968) Biochem. J. 108, 687-692. 8. Davis, B. J. (1964)Ann. N.Y. Acad. Sci. 121, 404-427. 9. Gabriel, 0. (1971) in “Methods in Enzymology” (Jakoby, W. B., ed.), Vol 12, pp. 565-578, Academic Press, New York.
