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Human plasma kallikrein
ARCHIVES
OF BIOCHEMISTRY
AND
165, 133-139 (1974)
BIOPHYSICS
Human Purification CLAUDIO Biology
SAMPAIO,Z Department,
Plasma
Kallikrein
and Preliminary SHOW-CHU
Brookhaoen
National
Received
April
Characterization’ WONG,
Laboratory,
AND
ELLIOTT
Upton,
New
York
SHAW 11973
15, 1974
A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-l. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esteraseand kinin-producing activities were enriched about llOO-fold from fraction IV-l. Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH,Cl was successful in contrast to the inertness of Tos-LysCH,Cl.
Kallikrein (EC 3.4.21.8) is the general designation for serine proteinases which have the ability to liberate physiologically active peptides called kinins from protein precursors by limited proteolysis (1). Proteases with this property have been found in urine, pancreatic secretions, and plasma but appear to be different entities (2). Nevertheless, they all have a specificity similar to trypsin in liberating kinins and may represent specialized proteases derived from the same ancestral gene possibly for a regulatory function in blood flow (3). To explore the relationship to trypsin further, we undertook the purification of the human plasma enzyme in the hope of obtaining a convenient supply. A number of methods have been described for the purification of kallikrein from human (4-6, 9), bovine (7), porcine (6, 8), and rabbit (9) plasma either as the active enzyme or the
inactive zymogen, prekallikrein. However, these generally involve many steps or provide a low yield. A relatively simple method was, therefore, devised which utilizes Cohn fraction IV-l of human plasma as starting material (5) and takes advantage of the affinity of kallikrein for soybean trypsin inhibitor (10) and for benzamidine (11). Adsorbents were prepared with these materials conjugated to Sepharose for use in affinity chromatography at successive steps of the isolation procedure. The use of such adsorbants for the purification of trypsin-like enzymes has been described (12-15) and applied to the purification of kallikrein (6). To activate prekallikrein, the plasma fraction was treated with acid (16) which presumably denatures inhibitors. Following this, kallikrein activity appeared spontaneously on incubation at neutrality, probably due to enzymic activation.
‘This work was supported by the U. S. Atomic Energy Commission and by U. S. Public Health Service Grant GM-17849. *Supported by a Public Health Service International Research Fellowship (No. TW 1645); present address: Department of Biochemistry, Escola Paulista Medicinea, Sao Paulo SPO4023, Brazil.
EXPERIMENTAL Materids. aminocaproic B Abbreviations 33
Copyright All rights
0 1974 hy Academic of reproduction
Press. Inc.
in any form
reserved.
PROCEDURE
Z-Lys-p-nitrophenyl acid, p-aminobenzamidine used:
Z-LysCH,Cl,
ester,3
the
N-c-Zdihydrochloro-
134
SAMPAIO,
WONG
chloride were from Cycle, benzamidine hydrochloride and I-cylohexyl-3-(2-morpholinoethyl)carbodiimide from Aldrich, cyanogen bromide and trifluoroacetic acid from Eastman, soybean trypsin inhibitor from Worthington, bradykinin from Schwarz-Mann, Sepharcee 48 and Sephadex derivatives from Pharmacia. Literature methods were used for the synthesis of Z-LysCH,Cl (17) and nitrophenyl p-guanidinobenzoate hydrochloride (18). Fraction IV-l of human plasma was very kindly provided by the Cutter Laboratories, Berkeley, CA, and arrived frozen with solid CO,. It was stored at -20°C until used. Soybean trypsin inhibitor conjugated to Sepharose was prepared from 500 mg of inhibitor and 200 ml of Sepharose 4B essentially as described by Feinstein (12) and equilibrated with 0.01 M phosphate buffer, pH 7.0, 0.15 M in NaCl. Preparation of the p-aminobenzamidine-Sepharose conjugate. p-(N- c-CbZ-amidocaproylamido) benzamidine hydrochloride was prepared by stirring a solution of p-aminobenzamidine. 2HCl (1.04 g). pyridine (2 ml), 1-cyclohexyl-3-(2-morpholinoethybcarbodiimide metho p-toluenesulfonate (2.33 g), and N-cCbZ-amidocaproic acid (1.33 g) in acetonitrile (30 ml) overnight. Following removal of the solvent, the residue was taken up in ethyl acetate and butanol (70 ml of 1: 1 mixture) and extracted with N HCl, water, 5% NaHCO,, and water. The dried organic layer, on concentration and treatment with ether provided 1.3 g of product, mp 176-177°C. This material (1.1 g) was deblocked by heating in trifluoroacetic acid (6 ml) at 90-95°C for 30 min. The residue was dissolved in excess methanolic HCl and taken up to dryness. The crude product was washed with ether and recrystallized from methanol and ether to yield 380 mg of p-(e-aminocaproylamido)benzamidine dihydrochloride, mp 284-285.5”C. Anal. Calcd for C,,H,,N,OCl,: C, 48.60; H, 6.90; N, 17.44. Found: C, 48,43; H, 6.89; N, 16.85. Sepharose 4B (30 ml) was activated with cyanogen bromide (7 g) in the usual way (19), suspended in 0.1 M NaHCO, buffer, pH 10.0 (20 ml), and stirred with a solution of p-(c-aminocaproylamido)benzamidine dihydrochloride (320 mg) in water (10 ml) which had been adjusted to pH 10.0. The mixture was maintained at 4°C for 24 hr and then washed thoroughly in a sintered glass funnel with water. It was stored in 0.75 M sodium chloride containing 0.02% sodium azide. The capacity of this material for kallikrein was about 100 units per ml.
methylketone derived from N”-benzyloxycarhonyl-Llysine; Tos-LysCH,Cl, the chloromethylketone derived from Nn-tosyl-n-lysine; abbreviations of substrate names conforms to recommended usage; STI, soybean trypsin inhibitor.
AND
SHAW
Biological assay. The assay of kallikrein by kinin release was observed through the use of the guinea pig ileum (20) in Tryode’s solution at 37°C measured with a Statham transducer and recorder. The substrate was freshly frozen human plasma heated 1 hr at 61°C. A dose-response curve was established with bradykinin as a reference standard. Enzyme assays. Esterase activity was measured with a pH-stat which recorded consumption of 0.01 M NaOH. The standard assay employed 4.0 ml of 0.02 M Tos-Arg-OMe in 0.06 M KC1 at pH 7.85, 25’C. The hydrolysis of 1 pmole of ester/min was considered to be 1 enzyme unit. Specific activity was defined as the ratio between enzyme units and absorbance at 280 nm. This assay was used to follow the purification procedure. Esterase activity was also examined spectroscopically on Z-Lys-ONp (21) in the concentration range 5.0 x 10m6-10m’ M in 0.2 M sodium maleate buffer, pH 6.0, and on nitrophenyl p-guanidinobenzoate at 10-5-10m’ M under conditions described for the titration of trypsin-like enzymes (18). Inhibition by benzamidine was evaluated by a Dixon plot of data obtained by examination of its effect on the esterase action of kallikrein on Z-LysONp (21). Electrophoresis. Disc gel electrophoresis was carried out by a literature method (22). Activation of prekallikrein. In a typical preparation, 1 kg of frozen fraction IV-l of human plasma was suspended in cold 0.06 M sodium chloride (3 liters) by brief treatment in a blender and centrifuged at 10,OOOg for 20 min at 4°C. The sediment was reextracted with an equivalent volume of cold saline and, following centrifugation, the supernatant solutions were combined and brought to a volume of 3200 ml. This solution, at about 10°C, was then slowly acidified with 6 N HCl to an apparent pH of 2.0 (16) and maintained there for 20 min with slow stirring at ambient temperature. The pH was then raised by the cautious addition of 6 N sodium hydroxide to 7.4 and the volume adjusted to 4 liters by the addition of 0.04 M phosphate buffer, pH 7.4, which was 0.6 M in sodium chloride. This solution was left at room temperature for l-2 hr to allow the activation of prekallikrein to proceed (Fig. 1). Esterase assays were carried out periodically until maximum activity was obtained and beginning to decline. Chromatography on STI-Sepharose. The activated plasma extract was slowly passed through a sintered glass funnel containing 206 ml of STI-Sepharose which had been previously equilibrated with 0.01 M sodium phosphate, pH 7.4, 0.15 M in sodium chloride. After the solution of kallikrein had passed through, the resin was extensively washed with the same phosphate buffer until the absorbance of the eluate at 280 nm dropped below 0.02. (About 8 liters of buffer was generally required.) The active material was then
HUMAN
PLASMA
KALLIKREIN
135
possible to follow the appearance of a new e&erase activity (Fig. 1). It was found possible to prepare kallikrein without acid treatment but the yield was much lower than in the procedure described above and the stability of the preparation was poorer. A part of the resultant esterase activity was not retained by STI-Sepharose. This generally amounted to 30-40% of the total initial activity (Table I). This material did not appear to be kallikrein since it was not inhibited by even a large excess of soybean trypsin inhibitor and did not liberate kinins from human plasma substrate as judged by biological assay. During the washing of the STI-Sepharose column with phosphate buffer, some esterase activity was eluted following the bulk protein peak. Since purification of the major bound esTIME (min) terase was found to be dependent on the extensive washing described, this small FIG. 1. Appearance of esterase activity in human loss of unidentified activity was not considplasma fraction IV-1 at pH 7.4 following exposure to ered important. A relatively high concenacid (30 min at pH 2.1). tration of benzamidine (about M) was eluted from the affinity adsorbant by four successive found to be necessary for the efficient washings with 250-ml portions of the above buffer, 1.0 elution of kallikrein from the STIM in benzamidine. HCI. The eluates were pooled and Sepharose column. dialyzed at 4°C with four changes of deionized water The benzamidine was largely removed (36 liters each) for about 40 hr followed by lyophilizaby dialysis, but not completely under the tion. The material was stored at -20°C. conditions described. Residual benzamiAffinity chromatography on the p-aminobenzamidine could be detected at 260 nm. Since it dine-Sepharose conjugate. Lyophilized product from appeared to have a beneficial effect on above (100 mg) was dissolved in 0.05 M phosphate stability as observed independently by buffer, pH 7.0, 0.75 M in NaCl (9 ml), and passed through a column (1.1 x 24 cm) of the conjugate others (6), complete removal was not atequilibrated in the same buffer. The column was tempted. However, the residual benzamiwashed with this buffer until no further protein was dine occasionally interfered with the rate eluted as judged by the absorbance at 280 nm assay, therefore for an accurate assessment whereupon 40 ml of the same buffer made M in of the progress of the purification, an benzamidine .HCl (and filtered) was applied. Twoaliquot of the preparation was gel filtered milliliter fractions were collected. The proteinand on Sephadex G-25 to permit a determinabenzamidine-containing fractions were pooled, passed tion of the specific activity (Table I). The through a Sephadex G-25 polystyrene column (2.0 x presence of a small amount of benzamidine 90 cm) equilibrated with 0.01 M ammonium acetate, may also have accounted for the stability of and eluted with the same buffer. the preparation to lyophilization. About RESULTS 90% of the activity could be recovered after The availability of human plasma fraclyophilization in contrast to the reported tion IV-l provided a favorable starting lability of prekallikrein (7) and kallikrein point for the isolation of kallikrein since it (5). Affinity chromatography on a phad been shown by Colman, Mattler, and aminobenzamidine Sepharose conjugate Sherry (5) that prekallikrein was concen- provided further purification (Table I). trated in this fraction. During the acid Elution was carried out with M benzamitreatment practically all of the initial es- dine as in the initial affinity chromatoterase activity disappeared. It was then graphic step.
136
SAMPAIO,
PURIFICATION
Step
Activation after acid Fraction not retained by STI-Sepharose column Benzamidine eluate of STI-Sepharose column after dialysis and lyophilization’ Fraction not retained by p-aminobenzamidine-Sepharose column Benzamidine eluate of paminobenzamidine-Sepharose column after Sephadex G-25 and lyophilization
OF HUMAN
Total protein (ODm)
197,000 172,000 533
341
92
WONG
AND
TABLE
I
PLASMA
KALLIKREIN
Total esterase activity
Specific
8,120 3,200 5,250
710
3,730
SHAW
FROM
activitya
COHN
FRACTION
IV-1
Purification
G-25 treated
Esterase activity
Biological activityb
0.04 0.02
-
-
-
9.9
34.5
860
840
2.5
-
-
-
-
45.1
1,130
1,200
(1)
(1)
% Total esterase activity recovered
(100) 64
-
46
a Specific activity = E.U./OD/ml; 1 enzyme unit (E.U.) = 1 pmole Tos-Arg-OMe hydrolyzed/min at pH 7.85. b Biological activity = pg bradykinin/OD/ml measured by guinea pig ileum bioassay. To remove kinins from the starting material (activated fraction IV-l), a sample was gel filtered (Sephadex G-25) at pH 4.5. ’ Benzamidine is not completely removed without G-25 treatment.
Although lower concentrations of benzamidine were also effective, larger volumes were required and the subsequent gel filtration become more cumbersome. The product at this point generally had a specific activity of 45-50 units per OD unit. Occasionally, samples with an activity above 60 were obtained. The reasons for this are not understood. The second affinity column thus provided further purification of the material concentrated by the initial, STI-Sepharose affinity column. In contrast, an attempt to use the arginine Sepharose conjugate described by Takahashi and colleagues (7) did not improve the purity of the STI-Sepharose product. Examination of this preparation on polyacrylamide gels in the native state revealed a single band (Fig. 2) which was the only region of the gel to contain esterase activity. In the presence of sodium dodecyl sulfate, multiple bands were found. Further investigation of this was deferred until final stages of purification were under study.
An apparent molecular weight of the active material determined by gel filtration on Sephadex G-200 was 95,000 (Fig. 3). The purification achieved from activated plasma was about llOO-fold if all of the initial esterase activity is considered to be kallikrein. However, this is not the case, since a portion of the initial activity was consistently not retained by the soybean trypsin inhibitor column; therefore, the actual purification of kallikrein must be greater. The biological (kinin-producing) activity was purified 1200-fold. Plasma kallikrein was found to be conveniently assayed spectroscopically through the use of Z-Lys-ONp for which a K, = 0.59 x 1O-4 was determined at pH 6. As expected, benzamidine was found to be a competitive inhibitor with Ki = 3.7 x lo-“. Nitrophenyl guanidinobenzoate was a suitable titrant for the enzyme since the nitrophenolate “burst” was constant in the reagent concentration range examined (10-4-10-5 h4), acylation was complete
137
HUMANPLASMAKALLIKREIN
enzymefromvariousspecies haveapproachedthis goalby purificationof the zymogen for subsequent activation by a plasma protease (7, 9). The alternate ap-
FIG. 2. Polyacrylamide disc-gel electrophoresis, pH 8.3, 0.01 M Tris-glycine, 23”C, 2 hr and 40 min at 2 mA/gel. Coomassie blue staining. Gel at right, product (0.2 mg) from STI-Sepharose column; gel at left, product (0.16 mg) from p-aminobenzoamidine-Sepharose conjugate.
within the few seconds required for mixing, and the deacylation rate for the acyl enzyme appeared to be quite low. The insensitivity of plasma kallikrein to Tos-LysCH,Cl was confirmed (23). However, another form of affinity label containing a lysine residue, namely, Z-LysCH&l, did inactivate the enzyme. At pH 7, for example, 4 x 1O-5 M Z-LysCH,Cl gave a half-time of inactivation of 15 min. From examination of the concentration dependence of the inactivation, it was determined that the reagent formed an intermediate complex with the enzyme with K, = 6.7 x lo-‘. DISCUSSION
Since plasma kallikrein occurs as a zymogen, certain recent efforts to isolate the
proach of activation at an early stage followed by purification has the attraction of convenience of assay, although in the case of proteolytic enzymes this may be offset by problems of autodigestion. This more direct approach has also been used with success in the purification of plasma kallikrein (5, 6, 9). The present work was designed to take advantage of the observation that plasma prekallikrein is concentrated in Cohn fraction IV-1 (5) whereas prothrombin and plasminogen are predominantly in other fractions (24). Since immobilized soybean trypsin inhibitor and benzamidine would also be expected to bind plasmin, thrombin, as well as other trypsin-like enzymes, this prior separation of the zymogens is a fortunate feature of the Cohn plasma fractionation scheme. Prekallikrein activator is also present in fraction IV-l apparently since spontaneous activation can be promoted on removal of an inhibitor by acidification. Colman et al. (5) found three forms of plasma kallikrein which they designated I, II, and III. The first two had molecular weights of 99,800 and 163,000, respectively, as judged by sedimentation equilibrium measurements, and were thought to exist possibly in a monomer-dimer relationship
100
t
\
FIG. 3. Estimation of molecular weight of human plasma kallikrein (HPK) by gel filtration on Sephadex G-200 (2 x 86 cm) in 0.01 M ammonium acetate, 0.1 M in NaCl, pH 6.0. (A) Chymotrypsinogen, (B) bovine serum albumin, (C) bovine serum albumin dimer.
138
SAMPAIO. WONG AND SHAW
(5). Together they constituted about 80% of the kallikrein activity in plasma. Since the minor form, kallikrein III, was poorly inhibited by soybean trypsin inhibitor (23), it would not be expected to be effectively retained by the Sepharose conjugate used to concentrate plasma kallikrein as described in the present work which led to a purified kallikrein of apparent M, = 95,000 probably corresponding to form I of Colman et al. Relatively similar molecular weights have been deduced by others (7,9) with some variation possibly due to species of origin and experimental method. Comparison of the specific activity obtained in the present work with that obtained by others is difficult because of the varied units employed by different authors. However, from published data (Ref. 5, Fig. 5) it may be calculated that the best preparation of Colman et al. had a specific activity of about 7 in the units employed in this paper and, therefore, was significantly less pure. The method described also is a relatively convenient source of kallikrein which appears to be pure as an esterase suitable for enzymatic studies involving the evaluation of inhibitors which was our initial goal. It also appears suitable as a source of pure enzyme for protein structural studies as will be subsequently described. Part of the success of the present method is due to the selectivity of the p-aminobenzamidine conjugate to Sepharose. Several other preparations and applications of this type of conjugate have been described (14, 15, 25). However, we believe that a significant difference in preparation exists between that described by us and others in that the weakly basic amino group of amino-benzamidine was coupled to tamino-caproic acid initially in the present work. Thus, the bond utilized in the attachment to Sepharose was the more reactive primary amino group of p-(taminocaproylamido)benzamidine. By contrast, attempts to couple carboxylic acid containing adsorbents to the amino group of p-amino-benzamidine as the final step invite a probably incomplete blockage of the carboxyl groups leading to an adsorbent with cation-exchange properties.
The relationship of plasma kallikrein to trypsin was strengthened by a number of observations such as the competitive inhibition by benzamidine (26) and the formation of a stable p-guanidinobenzoyl enzyme (18, 27). Related observations have been made with urinary and pancreatic kallikreins (11, 28-30) as well as the guinea pig plasma enzyme (31). The failure to be inactivated by the Tos-LysCH,Cl is apparently due to the tosyl group which may provide unfavorable interaction with the active center since Z-Lys CH,Cl was shown to be effective as are peptides terminating in LysCH,Cl (17). ACKNOWLEDGMENTS We are grateful to Dr. Duane Schroeder of the Cutter Laboratories for supplies of fraction IV-1 of human plasma and to Dr. L. J. Greene of this department for use of the equipment for pharmacological assay. Miss Janet Arnone provided considerable technical assistance in the purification procedure. In the early stages of this work information kindly provided by Dr. J. Pierce of the National Heart and Lung Institute was very helpful. REFERENCES 1. WEBSTER, M. E. (1970) in Handbuch der experimentellen Pharmakologie (Erdiis, E. G., ed.), Vol. 25, pp. 659-665, Springer-Verlag, New York. 2. WERLE, E. (1970) in Handbuch der experimentellen Pharmakologie (Erdos, E. G., ed.), Vol. 25, pp. 1-6, Springer-Verlag, New York. 3. SCHACHTER, M. (1969) Physiol. Reo. 49,509-547. 4. PIERCE, J. V. (1970) in Handbuch der experimentellen Pharmakologie (Erdos, E. G., ed.), Vol. 25, pp. 21-51, Springer-Verlag, New York. 5. COLMAN, R. W., MATTLER, L., AND SHERRY, S. (1969) J. Clin. Inuest. 48, 11-22. 6. FRITZ, H., WUNDERER, G., AND DI’ITMANN, B. (1972) Hoppe-Seyler’s 2. Physiol. Chem. 353, 893-900. 7. TAKAHASHI, H., NAGASAWA, S., AND SUZUKI, T. (1972) J. Biochem. 71, 471-483. 8. HABERMANN, E., AND KLETT, W. (1966) &o&em. 2. 346, 133-158. 9. WUEPPER, K. D., AND COCHRANE, C. G. (1972) J. Exp. Med. 135, l-20. 10. WERLE, E., ANDMAIER, L. (1952) Biochem. Z. 323, 279-283. 11. MARES-GUIA, M., AND DINE, C. R. (1967) Arch. Biochem. Biophys. 121.750-756. 12. FEINSTEIN, G. (1970) Biochim. Biophys. Acta 214, 224-227.
HUMAN
PLASMA
13. FEINSTEIN, G. (1970) Fed. EUF. Biochem. Sot. Lett. 7, 353-355. 14. SCHMER, G. (1972) 2. Physiol. Chem. 353, 810-814. 15. HIXSON, H. F., JR., AND NISHIKAWA, A. H. (1973) Arch. B&hem. Biophys. 154, 501-509. 16. KRAUT, H., FREY, E. K., AND WERLE, E. (1933) Hoppe-Seyler’s Z. Physiol. Chem. 222, 73-99. 17. COGGINS, J. R., Kray, W., AND SHAW, E. (1974) Biochem. J. 137, 579-585. 18. CHASE, T., JR., AND SHAW, E. (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 20-27, Academic Press, New York. 19. CUATRECASAS, P. (1970) J. Biol. Chem. 245, 3059-3065. 20. WEBSTER, M. E., AND PRADO, E. S. (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vo!. 19, pp. 68-699, Academic Press, New York. 21. WONG, S.-C., AND SHAW, E. (1974) Arch. Biochem. Biophys. 161, 536-543.
139
KALLIKREIN 22. ORNSTEIN, 321-349. 23. COLMAN,
L.
(1964)
R. W.,
Ann.
MATTLER,
N.
Y. Acad.
L.,
Sci.
AND SHERRY,
121,
S.
(1969) J. Clin. Inuest. 48, 23-32. 24. SCHULTZE, H. E., AND HEREMANS, J. F. (1966) Molecular Biology of Human Proteins, Vol. 1, pp. 250-252, Elsevier, New York. 25. JESTY, J., AND NEMERSON, Y. (1974) J. Biol. Chem. 249, 509-515. 26. MARES-GUIA, M., AND SHAW, E. (1965) J. Biol. Chem. 240, 1579-1585. 27. MARES-GUIA, M., AND SHAW, E. (1967) J. Biol. Chem. 242, 5782-5788. 28. FIEDLER, F., MULLER, B., AND WERLE, E. (1972) Fed. Eur. Biochem. Sot. Lett. 24, 41-44. 29. GERATZ, J. D. (1969) Experientia 25, 483-484. 30. MARKWARDT, F. (1970) Acta Biol. Med. Germ. 24, 401-404. 31. DAVIES, G. E., AND LOWE, J. S. (1970) in Bradykinin and Related Kinins (Sicuteri, F., Rocha e Silva, M., and Back, N., eds.), pp. 453-460, Plenum Press, New York.
OF BIOCHEMISTRY
AND
165, 133-139 (1974)
BIOPHYSICS
Human Purification CLAUDIO Biology
SAMPAIO,Z Department,
Plasma
Kallikrein
and Preliminary SHOW-CHU
Brookhaoen
National
Received
April
Characterization’ WONG,
Laboratory,
AND
ELLIOTT
Upton,
New
York
SHAW 11973
15, 1974
A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-l. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esteraseand kinin-producing activities were enriched about llOO-fold from fraction IV-l. Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH,Cl was successful in contrast to the inertness of Tos-LysCH,Cl.
Kallikrein (EC 3.4.21.8) is the general designation for serine proteinases which have the ability to liberate physiologically active peptides called kinins from protein precursors by limited proteolysis (1). Proteases with this property have been found in urine, pancreatic secretions, and plasma but appear to be different entities (2). Nevertheless, they all have a specificity similar to trypsin in liberating kinins and may represent specialized proteases derived from the same ancestral gene possibly for a regulatory function in blood flow (3). To explore the relationship to trypsin further, we undertook the purification of the human plasma enzyme in the hope of obtaining a convenient supply. A number of methods have been described for the purification of kallikrein from human (4-6, 9), bovine (7), porcine (6, 8), and rabbit (9) plasma either as the active enzyme or the
inactive zymogen, prekallikrein. However, these generally involve many steps or provide a low yield. A relatively simple method was, therefore, devised which utilizes Cohn fraction IV-l of human plasma as starting material (5) and takes advantage of the affinity of kallikrein for soybean trypsin inhibitor (10) and for benzamidine (11). Adsorbents were prepared with these materials conjugated to Sepharose for use in affinity chromatography at successive steps of the isolation procedure. The use of such adsorbants for the purification of trypsin-like enzymes has been described (12-15) and applied to the purification of kallikrein (6). To activate prekallikrein, the plasma fraction was treated with acid (16) which presumably denatures inhibitors. Following this, kallikrein activity appeared spontaneously on incubation at neutrality, probably due to enzymic activation.
‘This work was supported by the U. S. Atomic Energy Commission and by U. S. Public Health Service Grant GM-17849. *Supported by a Public Health Service International Research Fellowship (No. TW 1645); present address: Department of Biochemistry, Escola Paulista Medicinea, Sao Paulo SPO4023, Brazil.
EXPERIMENTAL Materids. aminocaproic B Abbreviations 33
Copyright All rights
0 1974 hy Academic of reproduction
Press. Inc.
in any form
reserved.
PROCEDURE
Z-Lys-p-nitrophenyl acid, p-aminobenzamidine used:
Z-LysCH,Cl,
ester,3
the
N-c-Zdihydrochloro-
134
SAMPAIO,
WONG
chloride were from Cycle, benzamidine hydrochloride and I-cylohexyl-3-(2-morpholinoethyl)carbodiimide from Aldrich, cyanogen bromide and trifluoroacetic acid from Eastman, soybean trypsin inhibitor from Worthington, bradykinin from Schwarz-Mann, Sepharcee 48 and Sephadex derivatives from Pharmacia. Literature methods were used for the synthesis of Z-LysCH,Cl (17) and nitrophenyl p-guanidinobenzoate hydrochloride (18). Fraction IV-l of human plasma was very kindly provided by the Cutter Laboratories, Berkeley, CA, and arrived frozen with solid CO,. It was stored at -20°C until used. Soybean trypsin inhibitor conjugated to Sepharose was prepared from 500 mg of inhibitor and 200 ml of Sepharose 4B essentially as described by Feinstein (12) and equilibrated with 0.01 M phosphate buffer, pH 7.0, 0.15 M in NaCl. Preparation of the p-aminobenzamidine-Sepharose conjugate. p-(N- c-CbZ-amidocaproylamido) benzamidine hydrochloride was prepared by stirring a solution of p-aminobenzamidine. 2HCl (1.04 g). pyridine (2 ml), 1-cyclohexyl-3-(2-morpholinoethybcarbodiimide metho p-toluenesulfonate (2.33 g), and N-cCbZ-amidocaproic acid (1.33 g) in acetonitrile (30 ml) overnight. Following removal of the solvent, the residue was taken up in ethyl acetate and butanol (70 ml of 1: 1 mixture) and extracted with N HCl, water, 5% NaHCO,, and water. The dried organic layer, on concentration and treatment with ether provided 1.3 g of product, mp 176-177°C. This material (1.1 g) was deblocked by heating in trifluoroacetic acid (6 ml) at 90-95°C for 30 min. The residue was dissolved in excess methanolic HCl and taken up to dryness. The crude product was washed with ether and recrystallized from methanol and ether to yield 380 mg of p-(e-aminocaproylamido)benzamidine dihydrochloride, mp 284-285.5”C. Anal. Calcd for C,,H,,N,OCl,: C, 48.60; H, 6.90; N, 17.44. Found: C, 48,43; H, 6.89; N, 16.85. Sepharose 4B (30 ml) was activated with cyanogen bromide (7 g) in the usual way (19), suspended in 0.1 M NaHCO, buffer, pH 10.0 (20 ml), and stirred with a solution of p-(c-aminocaproylamido)benzamidine dihydrochloride (320 mg) in water (10 ml) which had been adjusted to pH 10.0. The mixture was maintained at 4°C for 24 hr and then washed thoroughly in a sintered glass funnel with water. It was stored in 0.75 M sodium chloride containing 0.02% sodium azide. The capacity of this material for kallikrein was about 100 units per ml.
methylketone derived from N”-benzyloxycarhonyl-Llysine; Tos-LysCH,Cl, the chloromethylketone derived from Nn-tosyl-n-lysine; abbreviations of substrate names conforms to recommended usage; STI, soybean trypsin inhibitor.
AND
SHAW
Biological assay. The assay of kallikrein by kinin release was observed through the use of the guinea pig ileum (20) in Tryode’s solution at 37°C measured with a Statham transducer and recorder. The substrate was freshly frozen human plasma heated 1 hr at 61°C. A dose-response curve was established with bradykinin as a reference standard. Enzyme assays. Esterase activity was measured with a pH-stat which recorded consumption of 0.01 M NaOH. The standard assay employed 4.0 ml of 0.02 M Tos-Arg-OMe in 0.06 M KC1 at pH 7.85, 25’C. The hydrolysis of 1 pmole of ester/min was considered to be 1 enzyme unit. Specific activity was defined as the ratio between enzyme units and absorbance at 280 nm. This assay was used to follow the purification procedure. Esterase activity was also examined spectroscopically on Z-Lys-ONp (21) in the concentration range 5.0 x 10m6-10m’ M in 0.2 M sodium maleate buffer, pH 6.0, and on nitrophenyl p-guanidinobenzoate at 10-5-10m’ M under conditions described for the titration of trypsin-like enzymes (18). Inhibition by benzamidine was evaluated by a Dixon plot of data obtained by examination of its effect on the esterase action of kallikrein on Z-LysONp (21). Electrophoresis. Disc gel electrophoresis was carried out by a literature method (22). Activation of prekallikrein. In a typical preparation, 1 kg of frozen fraction IV-l of human plasma was suspended in cold 0.06 M sodium chloride (3 liters) by brief treatment in a blender and centrifuged at 10,OOOg for 20 min at 4°C. The sediment was reextracted with an equivalent volume of cold saline and, following centrifugation, the supernatant solutions were combined and brought to a volume of 3200 ml. This solution, at about 10°C, was then slowly acidified with 6 N HCl to an apparent pH of 2.0 (16) and maintained there for 20 min with slow stirring at ambient temperature. The pH was then raised by the cautious addition of 6 N sodium hydroxide to 7.4 and the volume adjusted to 4 liters by the addition of 0.04 M phosphate buffer, pH 7.4, which was 0.6 M in sodium chloride. This solution was left at room temperature for l-2 hr to allow the activation of prekallikrein to proceed (Fig. 1). Esterase assays were carried out periodically until maximum activity was obtained and beginning to decline. Chromatography on STI-Sepharose. The activated plasma extract was slowly passed through a sintered glass funnel containing 206 ml of STI-Sepharose which had been previously equilibrated with 0.01 M sodium phosphate, pH 7.4, 0.15 M in sodium chloride. After the solution of kallikrein had passed through, the resin was extensively washed with the same phosphate buffer until the absorbance of the eluate at 280 nm dropped below 0.02. (About 8 liters of buffer was generally required.) The active material was then
HUMAN
PLASMA
KALLIKREIN
135
possible to follow the appearance of a new e&erase activity (Fig. 1). It was found possible to prepare kallikrein without acid treatment but the yield was much lower than in the procedure described above and the stability of the preparation was poorer. A part of the resultant esterase activity was not retained by STI-Sepharose. This generally amounted to 30-40% of the total initial activity (Table I). This material did not appear to be kallikrein since it was not inhibited by even a large excess of soybean trypsin inhibitor and did not liberate kinins from human plasma substrate as judged by biological assay. During the washing of the STI-Sepharose column with phosphate buffer, some esterase activity was eluted following the bulk protein peak. Since purification of the major bound esTIME (min) terase was found to be dependent on the extensive washing described, this small FIG. 1. Appearance of esterase activity in human loss of unidentified activity was not considplasma fraction IV-1 at pH 7.4 following exposure to ered important. A relatively high concenacid (30 min at pH 2.1). tration of benzamidine (about M) was eluted from the affinity adsorbant by four successive found to be necessary for the efficient washings with 250-ml portions of the above buffer, 1.0 elution of kallikrein from the STIM in benzamidine. HCI. The eluates were pooled and Sepharose column. dialyzed at 4°C with four changes of deionized water The benzamidine was largely removed (36 liters each) for about 40 hr followed by lyophilizaby dialysis, but not completely under the tion. The material was stored at -20°C. conditions described. Residual benzamiAffinity chromatography on the p-aminobenzamidine could be detected at 260 nm. Since it dine-Sepharose conjugate. Lyophilized product from appeared to have a beneficial effect on above (100 mg) was dissolved in 0.05 M phosphate stability as observed independently by buffer, pH 7.0, 0.75 M in NaCl (9 ml), and passed through a column (1.1 x 24 cm) of the conjugate others (6), complete removal was not atequilibrated in the same buffer. The column was tempted. However, the residual benzamiwashed with this buffer until no further protein was dine occasionally interfered with the rate eluted as judged by the absorbance at 280 nm assay, therefore for an accurate assessment whereupon 40 ml of the same buffer made M in of the progress of the purification, an benzamidine .HCl (and filtered) was applied. Twoaliquot of the preparation was gel filtered milliliter fractions were collected. The proteinand on Sephadex G-25 to permit a determinabenzamidine-containing fractions were pooled, passed tion of the specific activity (Table I). The through a Sephadex G-25 polystyrene column (2.0 x presence of a small amount of benzamidine 90 cm) equilibrated with 0.01 M ammonium acetate, may also have accounted for the stability of and eluted with the same buffer. the preparation to lyophilization. About RESULTS 90% of the activity could be recovered after The availability of human plasma fraclyophilization in contrast to the reported tion IV-l provided a favorable starting lability of prekallikrein (7) and kallikrein point for the isolation of kallikrein since it (5). Affinity chromatography on a phad been shown by Colman, Mattler, and aminobenzamidine Sepharose conjugate Sherry (5) that prekallikrein was concen- provided further purification (Table I). trated in this fraction. During the acid Elution was carried out with M benzamitreatment practically all of the initial es- dine as in the initial affinity chromatoterase activity disappeared. It was then graphic step.
136
SAMPAIO,
PURIFICATION
Step
Activation after acid Fraction not retained by STI-Sepharose column Benzamidine eluate of STI-Sepharose column after dialysis and lyophilization’ Fraction not retained by p-aminobenzamidine-Sepharose column Benzamidine eluate of paminobenzamidine-Sepharose column after Sephadex G-25 and lyophilization
OF HUMAN
Total protein (ODm)
197,000 172,000 533
341
92
WONG
AND
TABLE
I
PLASMA
KALLIKREIN
Total esterase activity
Specific
8,120 3,200 5,250
710
3,730
SHAW
FROM
activitya
COHN
FRACTION
IV-1
Purification
G-25 treated
Esterase activity
Biological activityb
0.04 0.02
-
-
-
9.9
34.5
860
840
2.5
-
-
-
-
45.1
1,130
1,200
(1)
(1)
% Total esterase activity recovered
(100) 64
-
46
a Specific activity = E.U./OD/ml; 1 enzyme unit (E.U.) = 1 pmole Tos-Arg-OMe hydrolyzed/min at pH 7.85. b Biological activity = pg bradykinin/OD/ml measured by guinea pig ileum bioassay. To remove kinins from the starting material (activated fraction IV-l), a sample was gel filtered (Sephadex G-25) at pH 4.5. ’ Benzamidine is not completely removed without G-25 treatment.
Although lower concentrations of benzamidine were also effective, larger volumes were required and the subsequent gel filtration become more cumbersome. The product at this point generally had a specific activity of 45-50 units per OD unit. Occasionally, samples with an activity above 60 were obtained. The reasons for this are not understood. The second affinity column thus provided further purification of the material concentrated by the initial, STI-Sepharose affinity column. In contrast, an attempt to use the arginine Sepharose conjugate described by Takahashi and colleagues (7) did not improve the purity of the STI-Sepharose product. Examination of this preparation on polyacrylamide gels in the native state revealed a single band (Fig. 2) which was the only region of the gel to contain esterase activity. In the presence of sodium dodecyl sulfate, multiple bands were found. Further investigation of this was deferred until final stages of purification were under study.
An apparent molecular weight of the active material determined by gel filtration on Sephadex G-200 was 95,000 (Fig. 3). The purification achieved from activated plasma was about llOO-fold if all of the initial esterase activity is considered to be kallikrein. However, this is not the case, since a portion of the initial activity was consistently not retained by the soybean trypsin inhibitor column; therefore, the actual purification of kallikrein must be greater. The biological (kinin-producing) activity was purified 1200-fold. Plasma kallikrein was found to be conveniently assayed spectroscopically through the use of Z-Lys-ONp for which a K, = 0.59 x 1O-4 was determined at pH 6. As expected, benzamidine was found to be a competitive inhibitor with Ki = 3.7 x lo-“. Nitrophenyl guanidinobenzoate was a suitable titrant for the enzyme since the nitrophenolate “burst” was constant in the reagent concentration range examined (10-4-10-5 h4), acylation was complete
137
HUMANPLASMAKALLIKREIN
enzymefromvariousspecies haveapproachedthis goalby purificationof the zymogen for subsequent activation by a plasma protease (7, 9). The alternate ap-
FIG. 2. Polyacrylamide disc-gel electrophoresis, pH 8.3, 0.01 M Tris-glycine, 23”C, 2 hr and 40 min at 2 mA/gel. Coomassie blue staining. Gel at right, product (0.2 mg) from STI-Sepharose column; gel at left, product (0.16 mg) from p-aminobenzoamidine-Sepharose conjugate.
within the few seconds required for mixing, and the deacylation rate for the acyl enzyme appeared to be quite low. The insensitivity of plasma kallikrein to Tos-LysCH,Cl was confirmed (23). However, another form of affinity label containing a lysine residue, namely, Z-LysCH&l, did inactivate the enzyme. At pH 7, for example, 4 x 1O-5 M Z-LysCH,Cl gave a half-time of inactivation of 15 min. From examination of the concentration dependence of the inactivation, it was determined that the reagent formed an intermediate complex with the enzyme with K, = 6.7 x lo-‘. DISCUSSION
Since plasma kallikrein occurs as a zymogen, certain recent efforts to isolate the
proach of activation at an early stage followed by purification has the attraction of convenience of assay, although in the case of proteolytic enzymes this may be offset by problems of autodigestion. This more direct approach has also been used with success in the purification of plasma kallikrein (5, 6, 9). The present work was designed to take advantage of the observation that plasma prekallikrein is concentrated in Cohn fraction IV-1 (5) whereas prothrombin and plasminogen are predominantly in other fractions (24). Since immobilized soybean trypsin inhibitor and benzamidine would also be expected to bind plasmin, thrombin, as well as other trypsin-like enzymes, this prior separation of the zymogens is a fortunate feature of the Cohn plasma fractionation scheme. Prekallikrein activator is also present in fraction IV-l apparently since spontaneous activation can be promoted on removal of an inhibitor by acidification. Colman et al. (5) found three forms of plasma kallikrein which they designated I, II, and III. The first two had molecular weights of 99,800 and 163,000, respectively, as judged by sedimentation equilibrium measurements, and were thought to exist possibly in a monomer-dimer relationship
100
t
\
FIG. 3. Estimation of molecular weight of human plasma kallikrein (HPK) by gel filtration on Sephadex G-200 (2 x 86 cm) in 0.01 M ammonium acetate, 0.1 M in NaCl, pH 6.0. (A) Chymotrypsinogen, (B) bovine serum albumin, (C) bovine serum albumin dimer.
138
SAMPAIO. WONG AND SHAW
(5). Together they constituted about 80% of the kallikrein activity in plasma. Since the minor form, kallikrein III, was poorly inhibited by soybean trypsin inhibitor (23), it would not be expected to be effectively retained by the Sepharose conjugate used to concentrate plasma kallikrein as described in the present work which led to a purified kallikrein of apparent M, = 95,000 probably corresponding to form I of Colman et al. Relatively similar molecular weights have been deduced by others (7,9) with some variation possibly due to species of origin and experimental method. Comparison of the specific activity obtained in the present work with that obtained by others is difficult because of the varied units employed by different authors. However, from published data (Ref. 5, Fig. 5) it may be calculated that the best preparation of Colman et al. had a specific activity of about 7 in the units employed in this paper and, therefore, was significantly less pure. The method described also is a relatively convenient source of kallikrein which appears to be pure as an esterase suitable for enzymatic studies involving the evaluation of inhibitors which was our initial goal. It also appears suitable as a source of pure enzyme for protein structural studies as will be subsequently described. Part of the success of the present method is due to the selectivity of the p-aminobenzamidine conjugate to Sepharose. Several other preparations and applications of this type of conjugate have been described (14, 15, 25). However, we believe that a significant difference in preparation exists between that described by us and others in that the weakly basic amino group of amino-benzamidine was coupled to tamino-caproic acid initially in the present work. Thus, the bond utilized in the attachment to Sepharose was the more reactive primary amino group of p-(taminocaproylamido)benzamidine. By contrast, attempts to couple carboxylic acid containing adsorbents to the amino group of p-amino-benzamidine as the final step invite a probably incomplete blockage of the carboxyl groups leading to an adsorbent with cation-exchange properties.
The relationship of plasma kallikrein to trypsin was strengthened by a number of observations such as the competitive inhibition by benzamidine (26) and the formation of a stable p-guanidinobenzoyl enzyme (18, 27). Related observations have been made with urinary and pancreatic kallikreins (11, 28-30) as well as the guinea pig plasma enzyme (31). The failure to be inactivated by the Tos-LysCH,Cl is apparently due to the tosyl group which may provide unfavorable interaction with the active center since Z-Lys CH,Cl was shown to be effective as are peptides terminating in LysCH,Cl (17). ACKNOWLEDGMENTS We are grateful to Dr. Duane Schroeder of the Cutter Laboratories for supplies of fraction IV-1 of human plasma and to Dr. L. J. Greene of this department for use of the equipment for pharmacological assay. Miss Janet Arnone provided considerable technical assistance in the purification procedure. In the early stages of this work information kindly provided by Dr. J. Pierce of the National Heart and Lung Institute was very helpful. REFERENCES 1. WEBSTER, M. E. (1970) in Handbuch der experimentellen Pharmakologie (Erdiis, E. G., ed.), Vol. 25, pp. 659-665, Springer-Verlag, New York. 2. WERLE, E. (1970) in Handbuch der experimentellen Pharmakologie (Erdos, E. G., ed.), Vol. 25, pp. 1-6, Springer-Verlag, New York. 3. SCHACHTER, M. (1969) Physiol. Reo. 49,509-547. 4. PIERCE, J. V. (1970) in Handbuch der experimentellen Pharmakologie (Erdos, E. G., ed.), Vol. 25, pp. 21-51, Springer-Verlag, New York. 5. COLMAN, R. W., MATTLER, L., AND SHERRY, S. (1969) J. Clin. Inuest. 48, 11-22. 6. FRITZ, H., WUNDERER, G., AND DI’ITMANN, B. (1972) Hoppe-Seyler’s 2. Physiol. Chem. 353, 893-900. 7. TAKAHASHI, H., NAGASAWA, S., AND SUZUKI, T. (1972) J. Biochem. 71, 471-483. 8. HABERMANN, E., AND KLETT, W. (1966) &o&em. 2. 346, 133-158. 9. WUEPPER, K. D., AND COCHRANE, C. G. (1972) J. Exp. Med. 135, l-20. 10. WERLE, E., ANDMAIER, L. (1952) Biochem. Z. 323, 279-283. 11. MARES-GUIA, M., AND DINE, C. R. (1967) Arch. Biochem. Biophys. 121.750-756. 12. FEINSTEIN, G. (1970) Biochim. Biophys. Acta 214, 224-227.
HUMAN
PLASMA
13. FEINSTEIN, G. (1970) Fed. EUF. Biochem. Sot. Lett. 7, 353-355. 14. SCHMER, G. (1972) 2. Physiol. Chem. 353, 810-814. 15. HIXSON, H. F., JR., AND NISHIKAWA, A. H. (1973) Arch. B&hem. Biophys. 154, 501-509. 16. KRAUT, H., FREY, E. K., AND WERLE, E. (1933) Hoppe-Seyler’s Z. Physiol. Chem. 222, 73-99. 17. COGGINS, J. R., Kray, W., AND SHAW, E. (1974) Biochem. J. 137, 579-585. 18. CHASE, T., JR., AND SHAW, E. (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 20-27, Academic Press, New York. 19. CUATRECASAS, P. (1970) J. Biol. Chem. 245, 3059-3065. 20. WEBSTER, M. E., AND PRADO, E. S. (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vo!. 19, pp. 68-699, Academic Press, New York. 21. WONG, S.-C., AND SHAW, E. (1974) Arch. Biochem. Biophys. 161, 536-543.
139
KALLIKREIN 22. ORNSTEIN, 321-349. 23. COLMAN,
L.
(1964)
R. W.,
Ann.
MATTLER,
N.
Y. Acad.
L.,
Sci.
AND SHERRY,
121,
S.
(1969) J. Clin. Inuest. 48, 23-32. 24. SCHULTZE, H. E., AND HEREMANS, J. F. (1966) Molecular Biology of Human Proteins, Vol. 1, pp. 250-252, Elsevier, New York. 25. JESTY, J., AND NEMERSON, Y. (1974) J. Biol. Chem. 249, 509-515. 26. MARES-GUIA, M., AND SHAW, E. (1965) J. Biol. Chem. 240, 1579-1585. 27. MARES-GUIA, M., AND SHAW, E. (1967) J. Biol. Chem. 242, 5782-5788. 28. FIEDLER, F., MULLER, B., AND WERLE, E. (1972) Fed. Eur. Biochem. Sot. Lett. 24, 41-44. 29. GERATZ, J. D. (1969) Experientia 25, 483-484. 30. MARKWARDT, F. (1970) Acta Biol. Med. Germ. 24, 401-404. 31. DAVIES, G. E., AND LOWE, J. S. (1970) in Bradykinin and Related Kinins (Sicuteri, F., Rocha e Silva, M., and Back, N., eds.), pp. 453-460, Plenum Press, New York.