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The relationship between the lysine and the p-aminobenzamidine binding sites on human plasminogen
~HRO?IBOSIS RESEARCH Printed in the United
vol.
7,
pp.
Pergamon
States
683-693, Press,
1975 Inc.
THE RELATIONSHIP BETWEEN THE LYSINE AND THE p-AMINOBENZAMIDINE BINDING SITES ON HUMAN PLASMINOGEN
W.H. Holleman, W.W. Andres and L.J. Weiss Department of Biochemistry, Abbott Laboratories North Chicago, Illinois 60064
(Received
6.8.1975.
Accepted
by Editor
L. Lorand)
ABSTRACT With the use of affinity supports which were substituted with the ligands, lysine or p-aminobenzamidine, a site on human plasminogen was identified which bound p-aminobenzamidine, but was separate from the lysine binding site. This site was not the active site of the enzyme as inactivated plasmin also had a p-aminobenzamidine site. Benzamidine caused a conformational change in plasminogen which resulted in a decrease in the sedimentation coefficient from 5.1s to 4.6s. Methodology for the purification of gram quantities of human plasminogen is also discussed. INTRODUCTION Affinity chromatography on a support of lysine-agarose is the current method of choice for the purification of human plasminogen (1). Proper loading and washing of the lysine-agarose columns result in preparations which are at least 95% pure. The plasminogen is eluted from the column with E-aminocaproic acid (EACA), which is lysine minus the a-amino group. The plasminogens isolated in this manner have either Nterminal glutamic acid or N-terminal lysine depending on the starting material. Plasma and Cohn Fraction III yield plasminogens with N-terminal glutamic acid while Cohn Fraction III2,3 yields plasminogen with both Nterminal glutamic acid and N-terminal lysine (2). The N-terminal lysine species is the result of degradation of the N-terminal glutamic acid species by plasmin (3,4). As plasminogen is the inactive precursor of plasmin, it has no active site and therefore the binding of plasminogen to lysine-agarose must be at a site other than the active site. It has been shown by several investigators that the binding of lysine or EACA to plasminogen results in drastic changes in the sedimentation prop-
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erties of plasminogen, with the sedimentation coefficient decreasing from 5.1s to 4.3s (5,6). The stoichiometry of this interaction appears to be 1:l (7), with EACA being effective at one twentieth the concentration of lysine. The study reported here was initiated to further identify the lysine and/or EACA binding site in human plasminogen and resulted in the discovery of a site which binds p-aminobenzamidine, a compound which is a strong inhibitor of lysinelarginine esterolytic enzymes. These two sites appear to be located on different portions of the plasminogen molecule. MATERIALS AND METHODS Preparation of human plasminogen and plasmin Plasminogen was prepared from Cohn Fraction III2,3 by the affinity chromatography method of Deutsch and Mertz (1) as modified by Summaria using agarose substituted with 7 Pmole of lysine per ml of $t+;s{;' . The plasminogen was eluted with a linear gradient consisting of O-O.1 M EACA. This elution schedule resulted in the separation of the plasminogen into two fractions (Figure 1). The EACA concentrations in the peak tubes of Pools A and B were 0.010 M and 0.017 M respectively. After pooling of each peak the plasminogen was precipitated by adding 31 gm solid ammonium sulfate/100 ml. The solution was centrifuged and the precipitate was dissolved in the Tris-lysine buffer to give a protein concentration of 20 mg/ml. This solution was stored frozen until needed. The enzyme was assayed using the caseinolytic method of Robbins and Summaria (8). The plasminogen isolated in Pool B had an activity of 22-24 casein units per mg and was used for all of the experiments reported below. Plasmin was prepared by activation of plasminogen with small quantities of streptokinase (Kabikinase, Kabi AB) according to the method of Summaria -et al. (8). The plasmin prepared in this manner had an activity of 22-24 t 1 casein units per mg. p-aminobenzamidine-succinyl-diaminodipropylamino-agarose p-aminobenzamidine (Cycle Chemical Co.) was attached to Sepharose 4B with a spacer consisting of 3,3'-diaminodipropylamino-succinate. The methods for the reaction of 3,3'-diaminodipropylamine (Eastman Organics) with Sepharose 4B (Pharmacia, Inc.) followed by succinylation with succinic anhydride followed the procedures of Cuatracasas (9). This derivative was reacted with the ligand, p-aminobenzamidine? by suspending the derivatized agarose in 1 volume of distilled water, adding 5 g of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (Ott Chemical Co.) per 200 ml, titrating to pH 4.75 with 1 N HCl and adding 1 g of p-aminobenzamidine per 200 ml. The reaction was maintained at pH 4.75 and room temperature for 5 hours and then reacted overnight at 40. The derivatized agarose was washed sequentially with water, 1 M acetic acid, water, 0.01 M NaOH, water, 2 M NaCl and water. In order to obtain complete substitution of the succinyl groups with the ligand this reaction procedure was repeated 2 more times using one-half the amounts of the carbodiimide and p-aminobenzamidine. All of the free carboxyl groups were substituted with p-aminobenzamidine as measured by reaction of the derivative with glycine methyl ester according to the procedure of Hoare and Koshland (10). This final reacted agarose was suspended in 0.05 M Tris*HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 and stored
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at 4: p-nitrophenyl-p'-guanadinobenzoate (p-NPGB) titration of plasmin Titration of plasmin with p-NPGB was carried out according to the method of Chase and Shaw (11). The plasmin was diluted from 25% glycerol with 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 to give a protein concentration of 2-4 mg/ml. p-NPGB dissolved in DMF was added to the plasmin to give a 5-fold molar excess of p-NPGB. The reaction mixture was incubated at 250 for 15 min and the excess reagent removed by dialysis against several changes of the above buffer. Caseinolytic assay of the dialyzed plasmin showed that the inactivation process was 100% complete.
r
0
40
80
120 FRACTION
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240
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Figure 1 Purification of Human Plasminogen. Cohn Fraction III2,3 (2 kg) was extracted overnight at 4O with 12 1 of 0.05 M Tris, 0.02 M lysine, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 (Tris-lysine buffer). The insoluble material was centrifuged and the supernatant applied to a column (6.5 x 60 cm) of lysine-agarose which was equilibrated with~the Tris-lysine buffer. The column was washed with 0.05 M Tris*HCl, 0.5 M NaCl, 0.001 M EDTA, pH 9.0 (300 ml/h) until the A280 of the eluent was less than 0.1. At this point, a linear gradient consisting of 2 1 of 0.1 M sodium phosphate, pH 7.4 as the starting buffer and 2 1 of 0.1 M sodium phosphate, 0.1 M EACA, pH 7.4, as the limit buffer was passed over the column. Fractions of 16 ml were collected at 10 min intervals. This graph shows the absorbance profile after initiation of the EACA gradient.
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Affinity chromatography procedures All affinity column procedures with either lysine-agarose or with paminobenzamidine-succinyl-diaminodipropylamino-agarose (subsequently referred to as p-aminobenzamidine-agarose) were performed at 4O. The absorbed protein was eluted with either 0.2 M EACA or 0.2 M benzamidine.HCl (Aldrich Chemical Co.). When benzamidine was the eluent, the benzamidine was removed from the protein solution by dialysis at 40 against several changes of 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0. Specific details of the column operations are given in the figure legends. Sedimentation velocity Analytical sedimentation data were obtained using a Spinco Model E ultracentrifuge equipped with a split-beam automatic photoelectric scanning optical system. The sedimentation experiments were performed in 12 mm double sector cells at 60,000 rpm and at 20.0°. Plasminogen samples were prepared by dialyzing the diluted plasminogen (0.3 mg/ml) against 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 at 4O for 16 hours. After the completion of dialysis, benzamidine, dissolved in the above buffer, was added to each sample. These samples were incubated at room temperature for 30 minutes prior to the initiation of the sedimentation velocity experiment. RESULTS A 1 of the studies reported below used the plasminogen isolated from _. ___ . Pool B (see Figure 1). Application of this plasminogen to an affinity column composed of p-aminobenzamidine linked to agarose via a spacer of diaminodipropylaminosuccinate, resulted in complete binding of the plasminogen to the affinity resin. The plasminogen could be eluted in a small volume by washing the column with 0.2 M bentamidine, but attempts to elute the plasminogen with 0.2 M EACA were unsuccessful. As shown in Figure 1, this concentration of EACA was much greater than that necessary to elute plasminogen from lysine-agarose. The above results can be explained in two ways; 1) the sites on the plasminogen molecule responsible for binding lysine and p-aminobenzamidine are separate and independent of each other or, 2) there is one binding site for both ligands, but the binding of paminobenzamidine is stronger than the binding of lysine. If the latter is correct, EACA would not be expected to elute plasminogen from p-aminobenzamidine-agarose, but benzamidine would be expected to elute plasminogen from lysine-agarose. The experiments illustrated in Figure 2 suggest however that this is not the case, but rather that the two sites are separate and independent. In this experiment 0.2 M benzamidine did not elute the plasminogen from lysine-agarose while elution of the column with 0.2 M EACA resulted in an 85% recovery of the starting plasminogen. If, as the above described experiments indicate, plasminogen contains two binding sites, one specific for EACA and the second for p-aminobenzamidi.ne,it should be possible to bind plasminogen to lysine-agarose in the presence of benzamidine or conversely plasminogen should bind to p-aminobenzamidineagarose in the presence of EACA. The results of such experiments are shown in Figure 3. Figure 3a shows the results of the binding of plasminogen to a column of lysine-agarose which had been equilibrated with 0.2 M benzamidine. In addition the plasminogen solution contained 0.2 M benzamidine.
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60
NO
Figure 2 Absorption of plasminogen to lysine-agarose. 13.0 mg of plasminogen, dissolved in 4.3 ml of the Tris-lysine buffer, was applied to a 1.5 x 13 cm column of lysine-agarose which had been equilibrated at 4' with the above buffer. Fractions of 4 ml were collected at 6 min. intervals. The 0.05 M, 0.2 M benzamidine and 0.2 M EACA solutions were buffered at pH 7.4 with 0.1 M sodium phosphate.
The absorbance in fractions l-8 was due to the benzamidine in the column equilibration buffer. Elution of the lysine-Sepharose with a buffer containing 0.2 M EACA resulted in recovery of 92% of the applied plasminogen. Figure 3b illustrates the reverse experiment, where a plasminogen solution which contained 0.2 M EACA was absorbed to an affinity support of p-aminobenzamidine-Sepharose which had been equilibrated in the same buffer. Dialysis of the benzamidine containing fractions (28-45) resulted in recovery of 70% of the starting plasminogen. In a further attempt to characterize the p-aminobenzamidine binding site of plasminogen, the protein was reacted with the serine esterase inhibitor p-(m(m-fluoro-sulfonylphenylureido)phenoxyethoxy)benzamidine (FPPB). p-aminobenzamidine compounds are potent inhibitors of lysinelarginine esterases due to binding of the amidine function at the active site of the enzymes. Consequently FPPB binds to lysinelarginine esterases at the benzamidine end of the molecule leaving the sulfonylfluoride portion of the molecule free to react with a nucleophilic amino acid side chain located outside of the active site. The structure of the inhibitor is shown below
,(C"2)2-0
'S02-F
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Incubation of plasminogen with a lo-fold molar excess of FPPB resulted in the incorporation of 1.5 moles per mole of protein, but with only a 20% loss in caseinolytic activity. This modified protein was absorbed by both the p-aminobenzamidine and lysine affinity supports, indicating that the reaction between FPPB and plasminogen had not blocked either the p-aminobenzamidine or the EACA binding site. The results of the absorption and desorption of this derivatized plasminogen to lysine-sepharose are shown in Figure 4.
0 OSM 0 IM
lrir-HCI
0 OOIM
EDTA.
pH
9.0
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0.2M
BENZAMIDINE
EACA
1
i
??
2.0
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ai
N&I,
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Figure 3 (a). Absorption of plasminogen to lysine-agarose in the presence of benzamidine. Plasminogen (5.6 mg dissolved in 2.0 ml of 0.05 M Tris*HCl, 0.1 M NaCl, 0.001 M EDTA, 0.2 M benzamidine, pH 9.0) was applied to a 0.9 x 16 cm column of lysine-agarose which had been equilibrated in the above buffer. The Protein solution was washed in with 4 ml of the benzamidine containing buffer after which the column was washed with the same buffer, less the benzamidine, until the A2 was less than 0.05. At fraction 12, the plasminogen was eluted with 0.2 M !a CA buffered at pH 9.0 with 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA. Fractions of 8 ml were collected at 8 min intervals. (b). Absorption of plasminogen to p-aminobenzamidine-agarose in the presence of EACA. Plasminogen (5.7 mg in 2.5 ml) dissolved in 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA, 0.2 M EAcA, pH 9.0 was absorbed to a 0.9 x 16 cm column of p-aminobenzamidine-agarose. After washing the column with 60 ml of the loading buffer, the plasminogen was eluted with 0.2 M benzamidine buffered at PH 9.0 with 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA. Fractions 30-50 were pooled and exhaustively dialyzed to remove the benzamidine. Because p-aminobenzamidine is a potent inhibitor of plasmin it is possible, but unlikely, that the binding of the zymogen, Plasminogen, to the p-aminobenzamidine resin involves the potential active Site Of Plasminogen. To test this possibility plasmin was inactivated with the active Site titrant p-nitrophenyl-p'-guanadinobenzoate (NPGB). This plasmin, which was completely
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Figure 4
0.2M
EACA
.
10
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Absorption of FPPB-plasminogen to lysineagarose. FPPB (0.8 vq dissolved in 0.7 ml of DMSO) was added to 50 mg of plasminoqen (4.4 mg/ml) dissolved in the Tris-lysine buffer. After reaction at 37O for 90 min the excess reagent was removed by dialysis against 0.3 M glycine, pH 3.0. A control sample was also carried through the above procedure except that the FPPB was omitted. The dialyzed samples were subsequently dialyzed against the Trislysine buffer and their caseinolytic activities measured as described in methods. The FPPB-plasminogen (6.7 mg) was applied to a 1.5 x 16 cm column of lysine-agarose and washed with the iris-lysine buffer. The protein was eluted with 0.2 M EACA, 0.1 M phosphate, Na, pH 7.4. The flow rate of the column was 20 ml/h and fractions were collected every 12 min. 1 bl
4 IO
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EACA
0.2M
GRADIENT
BENZAMIDINE
c
1 08 A280 0.6
IO FRACTION
NO
20
40
30 FRACTION
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NO.
Figure 5 (a). Absorption of NPGB-plasmin to lysine-agarose. NPGB-plasmin (10.5 mg dissolved in 6.2 ml of the Tris-buffer) was applied to a 1.5 x 16 cm column of lysine-agarose. After washing the column with 50 ml of the above buffer the plasmin was eluted with an EACA gradient consisting of 50 ml of starting buffer (0.1 M phosphate, Na, pH 7.4) and 50 ml of limiting buffer (0.1 M phosphate, Na, 0.07 M EACA, pH 7.4). Fractions of 2 ml were collected at 6 min intervals. (b). Absorption of NPGB-plasmin to p-aminobenzamidine agarose. NPGB-plasmin (11.1 mg in 2.8 ml of the Tris buffer) was applied to a 0.9 x 20 cm column of p-aminobenzamidine-agarose. After washing with 60 ml of the loading buffer the column was eluted with 0.2 M benzamidine buffered at pH 9.0 with the Tris-buffer. Fractions 41-59 were pooled anddialyzed as described in methods.
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inactive towards casein, was indistinguishable from native plasminogen when absorbed to either lysine or p-aminobenzamidine-agarose. In both cases the inactivated plasmin was retained by tne column and in both cases the bulk of the activity was eluted by the competing ligand; EACA in the case of lysine-agarose (Figure 5a) and benzamidine in the case of p-aminobenzamidine-agarose (Figure 5b). The plasminogen which was not aosorbed to p-aminobenzamidine-agarose (Figure 5b) represented 15% ot the plasminogen applied to the column. Figure 6 shows the effect of increasing benzamidine concentrations on the sedimentation coefficient of plasminogen. The sedimentation coefficient of native plasminogen is reduced from 5.07s to 4.65s in the presence of 0.1 M benzamidine. Using the method of Castellino (5) and assuming that one mole of plasminogen binds one mole o1 benzamidine a KI for the binding of benzamidine to plasminogen of 2 x 10 M-1 was measured.
Figure 6 Sedimentation coefficient of plasminogen as a function of benzamidine concentration. See methods for experimental details.
Multiple molecular forms of human plasmino en with either NHp-terminal glutamic acid or NHp-terminal lysine (or valines have been described (2, 3, 4). When the isolation of plasminogen from human plasma is conducted in the presence of a protease inhibitor (e.g. Trasylol) only NH2-terminal glutamic acid plasminogen is obtained (4, 12). Incubation of this plasminogen with small amounts of plasmin result in the rapid conversion of the NH2-terminal glutamic acid species to the NH2-terminal lysine species (4, 13). Plasminogen isolated from Cohn Fraction 1112 3 the starting material used in these studies, in the absence of a proteAse inhibitor yields mainly NH2-terminal lysine plasminogen (2). This is not unexpected as the plasminogen which we purified from Cohn Fraction III2,3 always contained l-3% plasmin. If the
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plasminogen was eluted from the lysine-agarose column with 0.2 M EACA, rather than the EACA gradient used in Figure '1, the trailing portion of the peak contained large amounts of plasmin; the last few tubes containing as much as 50% plasmin. This finding suggests that plasmin binds more tightly to lysine-agarose than plasminogen. No attempts were made to measure the plasmin contents of the plasminogen fractions eluted from the lysine-agarose column by the EACA gradient. The results reported here (see Figure 1) show that the capacity of lysine-agarose for plasminogen is substantially greater than previously reported. Optimal binding of plasminogen was obtained when the ratio of Cohn Fraction III extract to lysine-agarose (v/v) was 6:l. Increasing the amount of Cohn fp3 raction 1112,3 extract actually resulted in a decrease in the amount of plasminogen recovered from the column. The elution profile shown in Figure 1 yielded 4720 mg of plasminogen (Pools A and B) or ca. 2.3 mg of plasminogen per ml of packed lysine-agarose. The separation of plasminogen into two distinct forms by the use of a gradient elution has been reported previously for rabbit plasminogen as well as for human plasminogen (5, 14). Castellino and coworkers, working with rabbit plasminogen, showed that these two forms have similar amino acid compositions, NH2-terminal amino acids and molecular weights, but vary in their sialic acid content. Collen has obtained similar results with the two subforms isolated from human plasma (14). Removal of the sialic acid from rabbit plasminogen did not change the binding of the two forms to lysine-agarose (15). The rabbit and human plasminogen used in the above studies contained NH2-terminal glutamic acid. The fact that we also seperated the plasminogen into two forms, using Cohn Fraction III2,3 as a starting material, suggest that the differences between the two subforms is not abolished by conversion of native plasminogen (NH2-terminal glutamic acid) to the altered form (NH2-terminal lysine). As plasminogen has no known enzymatic activity its binding to lysineagarose must be at a site distinct and separate from the active site. This conclusion is substantiated by the demonstration that plasmin whose active site has been titrated with p-NPGB will bind to lysine-agarose (Figure 4). Further evidence is that a complex of al-antitrypsin and plasmin has a higher affinity for lysine-agarose than the corresponding zymogen (16). The absorption of plasminogen to the p-aminobenzamidine-agarose affinity support was initially thought to have occurred at the lysine binding site. The failure of 0.5 M EACA to elute the protein was explained by assuming that the binding of p-aminobenzamidine to plasminogen was much stronger than its binding to lysine. Therefore the results shown in Figure 3 were unexpected. If lysine and p-aminobenzamidine were competing for a common site, plasminogen would not be expected to bind to lysine-agarose in the presence of benzamidine, as the benzamidine concentration (0.2 M) was ca. 30 times the lysine concentration (7 vmole/ml of agarose). Also if there was a single binding site which had a higher affinity for p-aminobenzamidine than lysine, benzamidine should have eluted the plasminogen which was absorbed to lysine-agarose. Figure 2 shows that 0.2 M benzamidine did not desorb the plasminogen from the lysine-agarose. The above experiments can be explained by postulating the presence of two binding sites on both the plasminogen and plasmin molecules; one site which binds lysine and a second site which binds p-aminobenzamidine.
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The exact role of the lysine binding site in plasminogen is unknown. However this site is apparently involved in the interaction between plasminogen and fibrin since the presence of EACA prevents the binding of plasminogen to fibrin (17, 18). EACA causes a large decrease in the sedimentation coefficient of plasminogen (5) and this conformational change may alter the interaction of plasminogen with fibrin. The p-aminobenzamidine site, presumably an arginine binding site in viva, may also be involved in this interaction between plasminogen and fibrin. Benzamidine, as does EACA, induces a conformational change in plasminogen as measured by a decrease in the sedimentation coefficient from 5.065 to 4.655 (Figure 6). This decrease is not as large as that induced by EACA where the sedimentation coeffficient drops from 5.1 to 4.3. Based on the assumption that one mole of pla mi ogen binds one mole of benzamidine, a dissociation constant of 2 x 10~,-~ is calculated. This value is approximately 5 fold greater than the KI (4.5 x 104M-1) calculated for the interaction of EACA with human plasminogen using the same technique (5). The sedimentation experiments were not repeated with p-aminobenzamidine but it can be assumed that p-aminobenzamidine would have an effect similar to benzamidine. REFERENCES 1.
DEUTSCH, D.G. and MERTZ, E.T. Plasminogen: purification from plasma by affinity chromatography. Science 170, 1095, 1970.
2.
SUMMARIA, L., ARZADON, L., BERNABE, P., and ROBBINS, K.C. Characterization of the NH2-terminal glutamic and NH2-terminal lysine forms of human plasminogen isolated by affinity chromatography and isoelectric focusing methods. J. Biol. Chem. 249, 2984, 1973.
3.
WALLEN, P. and WIMAN, B. Characterization of human plasminogen. I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations. Biochim. Biophys. Acta 221, 20, 1970.
4.
CLAEYS, H., MOLLA,_A., and VERSTRAETE, M. Conversion of NH2-terminal glutamic acid to NH2-terminal lysine human plasminogen by plasmin. Thrombosis Res. 3, 515, 1973.
5.
Measurement of the binding of BROCKWAY, W.J. and CASTELLINO, F.J., anfifibrinolytic amino acids to various plasminogens. Arch. Biochem. Biophys. 151, 194, 1972.
6.
ALKJAERSIG, N., FLETCHER, A.P. and SHERRY, S. e-aminocaproic acid: an inhibitor of plasminogen activation. J. Biol. Chem. 234, 832, 1959.
7.
ABIKO, Y., IWAMOTO, M., and TOMIKAWA, M. Plasminogen-plasmin system. V. A stoichiometric equilibrium complex of plasminogen and a synthetic inhibitor. Biochim. Biophys. Acta 185, 424, 1969.
8.
ROBBINS, K.C. and SUMMARIA, L. Human plasminogen and plasmin. Methods in Enzymol. 19, 184, 3970.
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CUATRECASAS, P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245, 3059, 1970.
10. HOARE, D.G. and KOSHLAND, D.E., JR. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. Biol. Chem. 242, 2447, 1967.
J :
11. CHASE, T., JR. and SHAW, E. Titration of trypsin, plasmin, and thrombin with p-nitrophenyl p'-guanidinobenzoate HCl. Methods in Enzymol. 19, 20, 1970. 12. WALLEN, P. and WIMAN,B. Characterization of human plasminogen. II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim. Biophys. Acta 257, 122, 1972. 13. SUMMARIA, L., ARZADON, L., BERNABE, P., and ROBBINS, K.C. The activation of plasminogen to plasmin by urokinase in the presence of the plasmin inhibitor Trasylol. J. Biol. Chem. 250, 3988, 1975. 14. COLLEN, D. (1973). De Microhetereogeneiteit von human plasminogen. Doctoraats proefschrift, Katholieke Universiteit, Leuvan, Belgium. 15. SIEFRING, G.E., JR. and CASTELLINO, F.J. The role of sialic acid in the determination of distinct properties of the isozymes of rabbit plasminogen. J. Biol. Chem. 249, 7742, 1974. 16. HATTON, M.W:C. and REGOECZI, E. Some observations on the affinity chromatography of rabbit plasminogen. Biochim. Biophys. Acta 359, 55, 1974. 17. THORSEN, S. Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation. Influence of w-aminocarboxylic acids. Biochim. Biophys. Acta 393, 55, 1975. 18. LANDMANN, H. Studies on the mechanism of action of synthetic antifibrinolytics. A comparison with the action of derivatives of benzamidine on the fibrinolytic process. Thrombos. Diathes. haemorrh. 29, 253, 1973.
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1975 Inc.
THE RELATIONSHIP BETWEEN THE LYSINE AND THE p-AMINOBENZAMIDINE BINDING SITES ON HUMAN PLASMINOGEN
W.H. Holleman, W.W. Andres and L.J. Weiss Department of Biochemistry, Abbott Laboratories North Chicago, Illinois 60064
(Received
6.8.1975.
Accepted
by Editor
L. Lorand)
ABSTRACT With the use of affinity supports which were substituted with the ligands, lysine or p-aminobenzamidine, a site on human plasminogen was identified which bound p-aminobenzamidine, but was separate from the lysine binding site. This site was not the active site of the enzyme as inactivated plasmin also had a p-aminobenzamidine site. Benzamidine caused a conformational change in plasminogen which resulted in a decrease in the sedimentation coefficient from 5.1s to 4.6s. Methodology for the purification of gram quantities of human plasminogen is also discussed. INTRODUCTION Affinity chromatography on a support of lysine-agarose is the current method of choice for the purification of human plasminogen (1). Proper loading and washing of the lysine-agarose columns result in preparations which are at least 95% pure. The plasminogen is eluted from the column with E-aminocaproic acid (EACA), which is lysine minus the a-amino group. The plasminogens isolated in this manner have either Nterminal glutamic acid or N-terminal lysine depending on the starting material. Plasma and Cohn Fraction III yield plasminogens with N-terminal glutamic acid while Cohn Fraction III2,3 yields plasminogen with both Nterminal glutamic acid and N-terminal lysine (2). The N-terminal lysine species is the result of degradation of the N-terminal glutamic acid species by plasmin (3,4). As plasminogen is the inactive precursor of plasmin, it has no active site and therefore the binding of plasminogen to lysine-agarose must be at a site other than the active site. It has been shown by several investigators that the binding of lysine or EACA to plasminogen results in drastic changes in the sedimentation prop-
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erties of plasminogen, with the sedimentation coefficient decreasing from 5.1s to 4.3s (5,6). The stoichiometry of this interaction appears to be 1:l (7), with EACA being effective at one twentieth the concentration of lysine. The study reported here was initiated to further identify the lysine and/or EACA binding site in human plasminogen and resulted in the discovery of a site which binds p-aminobenzamidine, a compound which is a strong inhibitor of lysinelarginine esterolytic enzymes. These two sites appear to be located on different portions of the plasminogen molecule. MATERIALS AND METHODS Preparation of human plasminogen and plasmin Plasminogen was prepared from Cohn Fraction III2,3 by the affinity chromatography method of Deutsch and Mertz (1) as modified by Summaria using agarose substituted with 7 Pmole of lysine per ml of $t+;s{;' . The plasminogen was eluted with a linear gradient consisting of O-O.1 M EACA. This elution schedule resulted in the separation of the plasminogen into two fractions (Figure 1). The EACA concentrations in the peak tubes of Pools A and B were 0.010 M and 0.017 M respectively. After pooling of each peak the plasminogen was precipitated by adding 31 gm solid ammonium sulfate/100 ml. The solution was centrifuged and the precipitate was dissolved in the Tris-lysine buffer to give a protein concentration of 20 mg/ml. This solution was stored frozen until needed. The enzyme was assayed using the caseinolytic method of Robbins and Summaria (8). The plasminogen isolated in Pool B had an activity of 22-24 casein units per mg and was used for all of the experiments reported below. Plasmin was prepared by activation of plasminogen with small quantities of streptokinase (Kabikinase, Kabi AB) according to the method of Summaria -et al. (8). The plasmin prepared in this manner had an activity of 22-24 t 1 casein units per mg. p-aminobenzamidine-succinyl-diaminodipropylamino-agarose p-aminobenzamidine (Cycle Chemical Co.) was attached to Sepharose 4B with a spacer consisting of 3,3'-diaminodipropylamino-succinate. The methods for the reaction of 3,3'-diaminodipropylamine (Eastman Organics) with Sepharose 4B (Pharmacia, Inc.) followed by succinylation with succinic anhydride followed the procedures of Cuatracasas (9). This derivative was reacted with the ligand, p-aminobenzamidine? by suspending the derivatized agarose in 1 volume of distilled water, adding 5 g of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (Ott Chemical Co.) per 200 ml, titrating to pH 4.75 with 1 N HCl and adding 1 g of p-aminobenzamidine per 200 ml. The reaction was maintained at pH 4.75 and room temperature for 5 hours and then reacted overnight at 40. The derivatized agarose was washed sequentially with water, 1 M acetic acid, water, 0.01 M NaOH, water, 2 M NaCl and water. In order to obtain complete substitution of the succinyl groups with the ligand this reaction procedure was repeated 2 more times using one-half the amounts of the carbodiimide and p-aminobenzamidine. All of the free carboxyl groups were substituted with p-aminobenzamidine as measured by reaction of the derivative with glycine methyl ester according to the procedure of Hoare and Koshland (10). This final reacted agarose was suspended in 0.05 M Tris*HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 and stored
w
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at 4: p-nitrophenyl-p'-guanadinobenzoate (p-NPGB) titration of plasmin Titration of plasmin with p-NPGB was carried out according to the method of Chase and Shaw (11). The plasmin was diluted from 25% glycerol with 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 to give a protein concentration of 2-4 mg/ml. p-NPGB dissolved in DMF was added to the plasmin to give a 5-fold molar excess of p-NPGB. The reaction mixture was incubated at 250 for 15 min and the excess reagent removed by dialysis against several changes of the above buffer. Caseinolytic assay of the dialyzed plasmin showed that the inactivation process was 100% complete.
r
0
40
80
120 FRACTION
160
200
240
NO.
Figure 1 Purification of Human Plasminogen. Cohn Fraction III2,3 (2 kg) was extracted overnight at 4O with 12 1 of 0.05 M Tris, 0.02 M lysine, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 (Tris-lysine buffer). The insoluble material was centrifuged and the supernatant applied to a column (6.5 x 60 cm) of lysine-agarose which was equilibrated with~the Tris-lysine buffer. The column was washed with 0.05 M Tris*HCl, 0.5 M NaCl, 0.001 M EDTA, pH 9.0 (300 ml/h) until the A280 of the eluent was less than 0.1. At this point, a linear gradient consisting of 2 1 of 0.1 M sodium phosphate, pH 7.4 as the starting buffer and 2 1 of 0.1 M sodium phosphate, 0.1 M EACA, pH 7.4, as the limit buffer was passed over the column. Fractions of 16 ml were collected at 10 min intervals. This graph shows the absorbance profile after initiation of the EACA gradient.
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Affinity chromatography procedures All affinity column procedures with either lysine-agarose or with paminobenzamidine-succinyl-diaminodipropylamino-agarose (subsequently referred to as p-aminobenzamidine-agarose) were performed at 4O. The absorbed protein was eluted with either 0.2 M EACA or 0.2 M benzamidine.HCl (Aldrich Chemical Co.). When benzamidine was the eluent, the benzamidine was removed from the protein solution by dialysis at 40 against several changes of 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0. Specific details of the column operations are given in the figure legends. Sedimentation velocity Analytical sedimentation data were obtained using a Spinco Model E ultracentrifuge equipped with a split-beam automatic photoelectric scanning optical system. The sedimentation experiments were performed in 12 mm double sector cells at 60,000 rpm and at 20.0°. Plasminogen samples were prepared by dialyzing the diluted plasminogen (0.3 mg/ml) against 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA, pH 9.0 at 4O for 16 hours. After the completion of dialysis, benzamidine, dissolved in the above buffer, was added to each sample. These samples were incubated at room temperature for 30 minutes prior to the initiation of the sedimentation velocity experiment. RESULTS A 1 of the studies reported below used the plasminogen isolated from _. ___ . Pool B (see Figure 1). Application of this plasminogen to an affinity column composed of p-aminobenzamidine linked to agarose via a spacer of diaminodipropylaminosuccinate, resulted in complete binding of the plasminogen to the affinity resin. The plasminogen could be eluted in a small volume by washing the column with 0.2 M bentamidine, but attempts to elute the plasminogen with 0.2 M EACA were unsuccessful. As shown in Figure 1, this concentration of EACA was much greater than that necessary to elute plasminogen from lysine-agarose. The above results can be explained in two ways; 1) the sites on the plasminogen molecule responsible for binding lysine and p-aminobenzamidine are separate and independent of each other or, 2) there is one binding site for both ligands, but the binding of paminobenzamidine is stronger than the binding of lysine. If the latter is correct, EACA would not be expected to elute plasminogen from p-aminobenzamidine-agarose, but benzamidine would be expected to elute plasminogen from lysine-agarose. The experiments illustrated in Figure 2 suggest however that this is not the case, but rather that the two sites are separate and independent. In this experiment 0.2 M benzamidine did not elute the plasminogen from lysine-agarose while elution of the column with 0.2 M EACA resulted in an 85% recovery of the starting plasminogen. If, as the above described experiments indicate, plasminogen contains two binding sites, one specific for EACA and the second for p-aminobenzamidi.ne,it should be possible to bind plasminogen to lysine-agarose in the presence of benzamidine or conversely plasminogen should bind to p-aminobenzamidineagarose in the presence of EACA. The results of such experiments are shown in Figure 3. Figure 3a shows the results of the binding of plasminogen to a column of lysine-agarose which had been equilibrated with 0.2 M benzamidine. In addition the plasminogen solution contained 0.2 M benzamidine.
1-01
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0
1
lbA 280 I2 -
a-
,
005 M
~
,
BENZAMIDINE I
I IO
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J 30 FRACTION
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50
60
NO
Figure 2 Absorption of plasminogen to lysine-agarose. 13.0 mg of plasminogen, dissolved in 4.3 ml of the Tris-lysine buffer, was applied to a 1.5 x 13 cm column of lysine-agarose which had been equilibrated at 4' with the above buffer. Fractions of 4 ml were collected at 6 min. intervals. The 0.05 M, 0.2 M benzamidine and 0.2 M EACA solutions were buffered at pH 7.4 with 0.1 M sodium phosphate.
The absorbance in fractions l-8 was due to the benzamidine in the column equilibration buffer. Elution of the lysine-Sepharose with a buffer containing 0.2 M EACA resulted in recovery of 92% of the applied plasminogen. Figure 3b illustrates the reverse experiment, where a plasminogen solution which contained 0.2 M EACA was absorbed to an affinity support of p-aminobenzamidine-Sepharose which had been equilibrated in the same buffer. Dialysis of the benzamidine containing fractions (28-45) resulted in recovery of 70% of the starting plasminogen. In a further attempt to characterize the p-aminobenzamidine binding site of plasminogen, the protein was reacted with the serine esterase inhibitor p-(m(m-fluoro-sulfonylphenylureido)phenoxyethoxy)benzamidine (FPPB). p-aminobenzamidine compounds are potent inhibitors of lysinelarginine esterases due to binding of the amidine function at the active site of the enzymes. Consequently FPPB binds to lysinelarginine esterases at the benzamidine end of the molecule leaving the sulfonylfluoride portion of the molecule free to react with a nucleophilic amino acid side chain located outside of the active site. The structure of the inhibitor is shown below
,(C"2)2-0
'S02-F
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Incubation of plasminogen with a lo-fold molar excess of FPPB resulted in the incorporation of 1.5 moles per mole of protein, but with only a 20% loss in caseinolytic activity. This modified protein was absorbed by both the p-aminobenzamidine and lysine affinity supports, indicating that the reaction between FPPB and plasminogen had not blocked either the p-aminobenzamidine or the EACA binding site. The results of the absorption and desorption of this derivatized plasminogen to lysine-sepharose are shown in Figure 4.
0 OSM 0 IM
lrir-HCI
0 OOIM
EDTA.
pH
9.0
0.2M
0.2M
BENZAMIDINE
EACA
1
i
??
2.0
b)
ai
N&I,
--
1.6 -
*DO 1.2
-
0.8
-
0.4
-
4
8
16
12 FRACTION
20
NO.
10
20
30 FRACTION
40
50
NO.
Figure 3 (a). Absorption of plasminogen to lysine-agarose in the presence of benzamidine. Plasminogen (5.6 mg dissolved in 2.0 ml of 0.05 M Tris*HCl, 0.1 M NaCl, 0.001 M EDTA, 0.2 M benzamidine, pH 9.0) was applied to a 0.9 x 16 cm column of lysine-agarose which had been equilibrated in the above buffer. The Protein solution was washed in with 4 ml of the benzamidine containing buffer after which the column was washed with the same buffer, less the benzamidine, until the A2 was less than 0.05. At fraction 12, the plasminogen was eluted with 0.2 M !a CA buffered at pH 9.0 with 0.05 M Tris.HCl, 0.1 M NaCl, 0.001 M EDTA. Fractions of 8 ml were collected at 8 min intervals. (b). Absorption of plasminogen to p-aminobenzamidine-agarose in the presence of EACA. Plasminogen (5.7 mg in 2.5 ml) dissolved in 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA, 0.2 M EAcA, pH 9.0 was absorbed to a 0.9 x 16 cm column of p-aminobenzamidine-agarose. After washing the column with 60 ml of the loading buffer, the plasminogen was eluted with 0.2 M benzamidine buffered at PH 9.0 with 0.05 M TrisaHCl, 0.1 M NaCl, 0.001 M EDTA. Fractions 30-50 were pooled and exhaustively dialyzed to remove the benzamidine. Because p-aminobenzamidine is a potent inhibitor of plasmin it is possible, but unlikely, that the binding of the zymogen, Plasminogen, to the p-aminobenzamidine resin involves the potential active Site Of Plasminogen. To test this possibility plasmin was inactivated with the active Site titrant p-nitrophenyl-p'-guanadinobenzoate (NPGB). This plasmin, which was completely
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Figure 4
0.2M
EACA
.
10
30
20
FRACTION
40 NO
50
Absorption of FPPB-plasminogen to lysineagarose. FPPB (0.8 vq dissolved in 0.7 ml of DMSO) was added to 50 mg of plasminoqen (4.4 mg/ml) dissolved in the Tris-lysine buffer. After reaction at 37O for 90 min the excess reagent was removed by dialysis against 0.3 M glycine, pH 3.0. A control sample was also carried through the above procedure except that the FPPB was omitted. The dialyzed samples were subsequently dialyzed against the Trislysine buffer and their caseinolytic activities measured as described in methods. The FPPB-plasminogen (6.7 mg) was applied to a 1.5 x 16 cm column of lysine-agarose and washed with the iris-lysine buffer. The protein was eluted with 0.2 M EACA, 0.1 M phosphate, Na, pH 7.4. The flow rate of the column was 20 ml/h and fractions were collected every 12 min. 1 bl
4 IO
O-O
07 M
EACA
0.2M
GRADIENT
BENZAMIDINE
c
1 08 A280 0.6
IO FRACTION
NO
20
40
30 FRACTION
50
NO.
Figure 5 (a). Absorption of NPGB-plasmin to lysine-agarose. NPGB-plasmin (10.5 mg dissolved in 6.2 ml of the Tris-buffer) was applied to a 1.5 x 16 cm column of lysine-agarose. After washing the column with 50 ml of the above buffer the plasmin was eluted with an EACA gradient consisting of 50 ml of starting buffer (0.1 M phosphate, Na, pH 7.4) and 50 ml of limiting buffer (0.1 M phosphate, Na, 0.07 M EACA, pH 7.4). Fractions of 2 ml were collected at 6 min intervals. (b). Absorption of NPGB-plasmin to p-aminobenzamidine agarose. NPGB-plasmin (11.1 mg in 2.8 ml of the Tris buffer) was applied to a 0.9 x 20 cm column of p-aminobenzamidine-agarose. After washing with 60 ml of the loading buffer the column was eluted with 0.2 M benzamidine buffered at pH 9.0 with the Tris-buffer. Fractions 41-59 were pooled anddialyzed as described in methods.
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inactive towards casein, was indistinguishable from native plasminogen when absorbed to either lysine or p-aminobenzamidine-agarose. In both cases the inactivated plasmin was retained by tne column and in both cases the bulk of the activity was eluted by the competing ligand; EACA in the case of lysine-agarose (Figure 5a) and benzamidine in the case of p-aminobenzamidine-agarose (Figure 5b). The plasminogen which was not aosorbed to p-aminobenzamidine-agarose (Figure 5b) represented 15% ot the plasminogen applied to the column. Figure 6 shows the effect of increasing benzamidine concentrations on the sedimentation coefficient of plasminogen. The sedimentation coefficient of native plasminogen is reduced from 5.07s to 4.65s in the presence of 0.1 M benzamidine. Using the method of Castellino (5) and assuming that one mole of plasminogen binds one mole o1 benzamidine a KI for the binding of benzamidine to plasminogen of 2 x 10 M-1 was measured.
Figure 6 Sedimentation coefficient of plasminogen as a function of benzamidine concentration. See methods for experimental details.
Multiple molecular forms of human plasmino en with either NHp-terminal glutamic acid or NHp-terminal lysine (or valines have been described (2, 3, 4). When the isolation of plasminogen from human plasma is conducted in the presence of a protease inhibitor (e.g. Trasylol) only NH2-terminal glutamic acid plasminogen is obtained (4, 12). Incubation of this plasminogen with small amounts of plasmin result in the rapid conversion of the NH2-terminal glutamic acid species to the NH2-terminal lysine species (4, 13). Plasminogen isolated from Cohn Fraction 1112 3 the starting material used in these studies, in the absence of a proteAse inhibitor yields mainly NH2-terminal lysine plasminogen (2). This is not unexpected as the plasminogen which we purified from Cohn Fraction III2,3 always contained l-3% plasmin. If the
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plasminogen was eluted from the lysine-agarose column with 0.2 M EACA, rather than the EACA gradient used in Figure '1, the trailing portion of the peak contained large amounts of plasmin; the last few tubes containing as much as 50% plasmin. This finding suggests that plasmin binds more tightly to lysine-agarose than plasminogen. No attempts were made to measure the plasmin contents of the plasminogen fractions eluted from the lysine-agarose column by the EACA gradient. The results reported here (see Figure 1) show that the capacity of lysine-agarose for plasminogen is substantially greater than previously reported. Optimal binding of plasminogen was obtained when the ratio of Cohn Fraction III extract to lysine-agarose (v/v) was 6:l. Increasing the amount of Cohn fp3 raction 1112,3 extract actually resulted in a decrease in the amount of plasminogen recovered from the column. The elution profile shown in Figure 1 yielded 4720 mg of plasminogen (Pools A and B) or ca. 2.3 mg of plasminogen per ml of packed lysine-agarose. The separation of plasminogen into two distinct forms by the use of a gradient elution has been reported previously for rabbit plasminogen as well as for human plasminogen (5, 14). Castellino and coworkers, working with rabbit plasminogen, showed that these two forms have similar amino acid compositions, NH2-terminal amino acids and molecular weights, but vary in their sialic acid content. Collen has obtained similar results with the two subforms isolated from human plasma (14). Removal of the sialic acid from rabbit plasminogen did not change the binding of the two forms to lysine-agarose (15). The rabbit and human plasminogen used in the above studies contained NH2-terminal glutamic acid. The fact that we also seperated the plasminogen into two forms, using Cohn Fraction III2,3 as a starting material, suggest that the differences between the two subforms is not abolished by conversion of native plasminogen (NH2-terminal glutamic acid) to the altered form (NH2-terminal lysine). As plasminogen has no known enzymatic activity its binding to lysineagarose must be at a site distinct and separate from the active site. This conclusion is substantiated by the demonstration that plasmin whose active site has been titrated with p-NPGB will bind to lysine-agarose (Figure 4). Further evidence is that a complex of al-antitrypsin and plasmin has a higher affinity for lysine-agarose than the corresponding zymogen (16). The absorption of plasminogen to the p-aminobenzamidine-agarose affinity support was initially thought to have occurred at the lysine binding site. The failure of 0.5 M EACA to elute the protein was explained by assuming that the binding of p-aminobenzamidine to plasminogen was much stronger than its binding to lysine. Therefore the results shown in Figure 3 were unexpected. If lysine and p-aminobenzamidine were competing for a common site, plasminogen would not be expected to bind to lysine-agarose in the presence of benzamidine, as the benzamidine concentration (0.2 M) was ca. 30 times the lysine concentration (7 vmole/ml of agarose). Also if there was a single binding site which had a higher affinity for p-aminobenzamidine than lysine, benzamidine should have eluted the plasminogen which was absorbed to lysine-agarose. Figure 2 shows that 0.2 M benzamidine did not desorb the plasminogen from the lysine-agarose. The above experiments can be explained by postulating the presence of two binding sites on both the plasminogen and plasmin molecules; one site which binds lysine and a second site which binds p-aminobenzamidine.
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The exact role of the lysine binding site in plasminogen is unknown. However this site is apparently involved in the interaction between plasminogen and fibrin since the presence of EACA prevents the binding of plasminogen to fibrin (17, 18). EACA causes a large decrease in the sedimentation coefficient of plasminogen (5) and this conformational change may alter the interaction of plasminogen with fibrin. The p-aminobenzamidine site, presumably an arginine binding site in viva, may also be involved in this interaction between plasminogen and fibrin. Benzamidine, as does EACA, induces a conformational change in plasminogen as measured by a decrease in the sedimentation coefficient from 5.065 to 4.655 (Figure 6). This decrease is not as large as that induced by EACA where the sedimentation coeffficient drops from 5.1 to 4.3. Based on the assumption that one mole of pla mi ogen binds one mole of benzamidine, a dissociation constant of 2 x 10~,-~ is calculated. This value is approximately 5 fold greater than the KI (4.5 x 104M-1) calculated for the interaction of EACA with human plasminogen using the same technique (5). The sedimentation experiments were not repeated with p-aminobenzamidine but it can be assumed that p-aminobenzamidine would have an effect similar to benzamidine. REFERENCES 1.
DEUTSCH, D.G. and MERTZ, E.T. Plasminogen: purification from plasma by affinity chromatography. Science 170, 1095, 1970.
2.
SUMMARIA, L., ARZADON, L., BERNABE, P., and ROBBINS, K.C. Characterization of the NH2-terminal glutamic and NH2-terminal lysine forms of human plasminogen isolated by affinity chromatography and isoelectric focusing methods. J. Biol. Chem. 249, 2984, 1973.
3.
WALLEN, P. and WIMAN, B. Characterization of human plasminogen. I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations. Biochim. Biophys. Acta 221, 20, 1970.
4.
CLAEYS, H., MOLLA,_A., and VERSTRAETE, M. Conversion of NH2-terminal glutamic acid to NH2-terminal lysine human plasminogen by plasmin. Thrombosis Res. 3, 515, 1973.
5.
Measurement of the binding of BROCKWAY, W.J. and CASTELLINO, F.J., anfifibrinolytic amino acids to various plasminogens. Arch. Biochem. Biophys. 151, 194, 1972.
6.
ALKJAERSIG, N., FLETCHER, A.P. and SHERRY, S. e-aminocaproic acid: an inhibitor of plasminogen activation. J. Biol. Chem. 234, 832, 1959.
7.
ABIKO, Y., IWAMOTO, M., and TOMIKAWA, M. Plasminogen-plasmin system. V. A stoichiometric equilibrium complex of plasminogen and a synthetic inhibitor. Biochim. Biophys. Acta 185, 424, 1969.
8.
ROBBINS, K.C. and SUMMARIA, L. Human plasminogen and plasmin. Methods in Enzymol. 19, 184, 3970.
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9.
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CUATRECASAS, P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245, 3059, 1970.
10. HOARE, D.G. and KOSHLAND, D.E., JR. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. Biol. Chem. 242, 2447, 1967.
J :
11. CHASE, T., JR. and SHAW, E. Titration of trypsin, plasmin, and thrombin with p-nitrophenyl p'-guanidinobenzoate HCl. Methods in Enzymol. 19, 20, 1970. 12. WALLEN, P. and WIMAN,B. Characterization of human plasminogen. II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim. Biophys. Acta 257, 122, 1972. 13. SUMMARIA, L., ARZADON, L., BERNABE, P., and ROBBINS, K.C. The activation of plasminogen to plasmin by urokinase in the presence of the plasmin inhibitor Trasylol. J. Biol. Chem. 250, 3988, 1975. 14. COLLEN, D. (1973). De Microhetereogeneiteit von human plasminogen. Doctoraats proefschrift, Katholieke Universiteit, Leuvan, Belgium. 15. SIEFRING, G.E., JR. and CASTELLINO, F.J. The role of sialic acid in the determination of distinct properties of the isozymes of rabbit plasminogen. J. Biol. Chem. 249, 7742, 1974. 16. HATTON, M.W:C. and REGOECZI, E. Some observations on the affinity chromatography of rabbit plasminogen. Biochim. Biophys. Acta 359, 55, 1974. 17. THORSEN, S. Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation. Influence of w-aminocarboxylic acids. Biochim. Biophys. Acta 393, 55, 1975. 18. LANDMANN, H. Studies on the mechanism of action of synthetic antifibrinolytics. A comparison with the action of derivatives of benzamidine on the fibrinolytic process. Thrombos. Diathes. haemorrh. 29, 253, 1973.