t u r . J. Biochem. 5Y. 441 -447 (1975)
A New Method of Isolation and Some Properties of the Heavy Chain of Human Plasmin Egon E. RICKLI and Wendy I. OTAVSKY Theodor Kocher Institute and Institute of Organic Chemistry, University of Berne (Received October 8, 1974 / August 2. 1975)
A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60- 65 O b ) . Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH,-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
The results from different laboratories have shown that the molecular structures of plasminogen and plasrnin are distinctly different. Plasminogen consists of a single polypeptide chain [ 1 - 31, which is selectively cleaved during the activation process to finally yield the plasmin molecule composed of two polypeptide chains. The two chain structure of plasmin was originally observed by Robbins et al. [l] who reported during activation with urokinase the cleavage of a single arginylvalyl bond in plasminogen, thereby producing a heavy (A) and a light (B) chain which in the native plasmin molecule are apparently connected by a single disulfide bond. The individual chains were demonstrated by starch gel electrophoresis of reduced and carboxymethylated plasmin [l]. The two chain structure of human plasmin was recently confirmed by Wiman and Wallen [4] and also by preliminary results of Rickli and Otavsky [ 5 ] . The isolation and preparation of the individual chains of plasmin first implies the cleavage of the interchain disulfide linkage. Robbins et al. [I] obtained a seemingly preferential cleavage of the interchain disulfide bridge by reduction with 2-mercaptoethanol at Abbreviurion. Dansyl, 5-dimethylaminonaphthalene-1-sulphonyl. Enzyme. Plasmin (EC 3.4.21.7).
low concentration in the presence of Tris buffer, pH 9 in 8 M urea. The cleavage of the intrachain disulfide bonds at alkaline pH apparently requires somewhat stronger reducing conditions. A separation of the two chains of plasmin was achieved by different methods during which extensive reduction and carboxymethylation were usually employed. Essentially pure light and heavy chain preparations were obtained by Wiman and Wallen [4] by gel filtration of the reduced and carboxymethylated plasmin on Sephadex G-150 in 0.1 M Tris, 6 M urea, pH 7.6. Summaria and Robbins [6] prepared purified heavy chain by first dialyzing completely reduced and carboxymethylated plasmin against 0.002 M ammonium bicarbonate thus precipitating most of the light chain. Contaminating amounts of the fragment in the soluble heavy chain fraction were then eliminated by precipitation with 1 M trichloroacetic acid in 8 M urea. In the present study we described a new and simple method for the preparation of the heavy chain of human plasmin. This procedure is based on the ability of the heavy chain fragment, obtained by mild reduction of plasmin, to combine with the adsorbent polyacrylamide-lysine used by us for the isolation of plasminogen from plasma. In addition, we report some properties of the purified heavy chain which, in contrast to other known preparations, has most of its disulfide bonds intact.
Heavy Chain of Human Plasmin
442
MATERIALS AND METHODS Plasminogen
Plasminogen was isolated from 500 - 1000 ml pooled fresh, human ACD-plasma by affinity chromatography as previously published [3]. The elution of the proenzyme from the affinity adsorbent was achieved with 0.3 M phosphate buffer, containing 0.2 M 6-aminohexanoic acid, pH 7.4. Plasminogen was precipitated from the pooled protein-containing fractions with 2 M (NHJ2S04 at pH 7.4, and dialyzed at 2°C for at least 36 h against several changes of 0.05 M Tris-buffer, containing 0.1 M NaC1, pH 7.8 or 9.0. This plasminogen was characterized by glutamic acid as the sole NH,-terminal amino acid residue. The preparations were free of any detectable spontaneous plasmin activity as determined by a caseinolytic assay. The specific activity after activation with streptokinase [3] was between 22 and 27U/mg protein. This is less than previously published [3], and is probably due to larger batches which require a longer time for the completion of the isolation process. Activation of Plasminogen with Urokinase
For the activation of plasminogen the procedure of Robbins et al. [7] was applied in a slightly modified form. Plasminogen was used in a concentration of about 5 mg/ml in 0.05 M Tris-buffer, containing 0.1 M NaCl, pH 7.8, and 25% (v/v) glycerol. Urokinase (generously supplied as “urokinase reagent” [approx. 20 000 units/vial] by Dr R. Strassle, Hoffmann-La Roche, Basel) was then added in a ratio of 1 unit per unit of plasminogen and the activation was allowed to proceed at room temperature for 8 h at which time an additional 0.5 units of urokinase per unit plasminogen was added. Activation was continued for a total of 24 h. NH2-Terminal Amino Acid Analysis
NH,-terminal analyses were carried out with the fluordinitrobenzene method of Sanger [8] in the presence of 8 M urea, and the dinitrophenyl amino acids were analyzed by two-dimensional thin-layer chromatography on silica gel G. In the first dimension the dimension the “toluene solvent system” [9] was used; the solvent of the second dimension was chloroform/ benzyl alcohol/acetic acid (70/30/3, by vol.) [lo]. The results of this method were verified with the dansyl technique. The dansylation procedure of Gros and Labouesse [ l l ] as well as that of Gray [12] was used. After acid hydrolysis at 105- 110 “C for 12- 15 h the samples were dried in V ~ C U Oat 40°C and taken up in 20 - 25 pl of 50 pyridine. For two-dimensional thinlayer chromatography samples of approx. 1 p1 were applied on polyamide sheets (Schleicher & Schull F 1700) coated on both sides. On the reverse side of the
sheets a mixture of standard dansyl amino acids was spotted in the same position as the unknown sample. For the development of the chromatograms the solvent systems of Woods and Wang [13] were used with water/90 % formic acid (200/3, v/v) in the first dimension and benzene/acetic acid (9/1, v/v) in the second dimension. The chromatograms were re-run in the second dimension in ethyl acetate/methanol/acetic acid (20/1/1, by vol.) [14]. Polyacrylamide Electrophoresis
Dodecylsulfate electrophoresis on polyacrylamide gel was camed out according to Weber and Osborn [15]. Gels made from 10% acrylamide and 0.27g of methylene bisacrylamide in 0.1 M phosphate buffer, pH 7.0, containing 0.1 % sodium dodecylsulfate were used. The protein (approx. 0.5 mg/ml) was treated for 2 h at 37 “C with 0.01 M phosphate buffer, pH7.0, containing 1 %, sodium dodecylsulfate and 1 % 2-mercaptoethanol. Samples of 20- 50 pg of protein were applied per gel column (6 x 100mm) and electrophoresis was performed at 8 volts per gel for 6 h after which the gels were stained with Coomassie brilliant blue. For the estimation of molecular weights the following reference proteins were used : human transferrin, bovine glutamate dehydrogenase, hog pepsin, bovine a-chymotrypsinogen A (all purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A.)and human serum albumin (supplied by the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross, Berne). Uliracentrifugal Analyses
Sedimentation and diffusion measurements were carried out in an analytical Spinco, model E, ultracentrifuge at 20 “C. The protein (4 mg/ml) to be analyzed was exhaustively dialyzed at 2 “C against 0.05 M Tris/HCI buffer, pH 7.8, containing 0.1 M NaCl. For one series of measurements this buffer system contained in addition 6-aminohexanoic acid in concentrations varying between 0.1 M and 1 pM. The addition of 6aminohexanoic acid to the buffer always took place before the final pH-adjustment. Sedimentationanalyses were performed in a standard 12 mm cell at 59 780 rev./ min. Diffusion measurements were made in a synthetic boundary cell (valve type, 12 mm) at a speed of 4609 rev./min. Titration of Sulfhydryl Groups
Free sulfhydryl groups were determined according to the spectrophotometric titration method of Boyer [16]. The protein, usually in a concentration of about 4 mg/ml, was titrated in 0.33 M sodium acetate, pH 4.6, containing 8 M urea with p-chloromercuribenzoate
443
E. E. Rickli and W.1. Otdvsky
(Calbiochem) which had been purified by repeated acid precipitations from alkaline solution as recommended by Boyer [16]. Some determinations were also made in the absence of urea. A chloromercuribenzoate solution of approximately 0.6 mM in water (solubilized by a few drops of 0.05 N NaOH) was used. The exact concentration was determined by absorbance measurements at 232 nm at pH 7, using the molar absorption coefficient of 16 900 M-' cm-' as given by Boyer [16]. The titrations were carried out in a Beckman, model DB, spectrophotometer at 225 nm with 3.0 ml of protein solution of known concentration in both the reference and sample cells. To the sample cell were added constant amounts (usually 20 pl) of the chloromercuribenzoate solution. After each addition of titrant the contents of the cell were well mixed and the increase in absorbance was recorded. The absorbance values (corrected for dilution) were then plotted as a function of the chloromercuribenzoate concentration. This resulted in two straight lines with different slopes. The intersection point of the two lines was considered to be the equivalence point of the titration. From the amount of titrant consumed at the equivalence point the amount of free sulfhydryls was calculated.
Preparation and Isolation of the Plasmin Heavy Chain
Plasminogen was activated with urokinase for 24 h as described above. The activated sample was reduced by treatment with 0.1 M 2-mercaptoethanol for 20 min at 20 "C under nitrogen. The mixture was then cooled immediately in ice and applied without delay on a column filled with lysine-substituted polyacrylamide (Biogel P-300), equilibrated in the cold against 0.05 M phosphate buffer, containing 0.1 M NaC1, pH 7.4. The amount of polyacrylamide-lysine was approx. 2 ml of packed gel per mg plasminogen activated. In typical experiments plasminogen in amounts between 60 and 80 mg was used. To insure adequate flow rates acolumn with a cross section of about 20 cm2was chosen. A constant flow rate of 27.2 ml/h was provided with a perspex pump. The column effluent was collected in 5 ml fractions, and the protein content of the fractions was determined from absorbance measurements at 280 nm. The buffers were prepared with boiled water and kept in closed containers under nitrogen. After the protein solution had penetrated into the gel bed the chromatogram was developed by washing the column with 0.05 M phosphate buffer, containing 0.1 M NaCl, pH 7.4. This resulted in the displacement of unadsorbed protein and low molecular weight compounds of the reaction mixture which emerged from the column in a first major peak (fraction I) (Fig. 1). Washing was continued until the absorbance of the effluent had decreased below 0.03. At this point the
Fraction number
Fig. 1. Isolation of the plasmin heavy chain on a column oj'polyacrylamide- lysine. Fraction I represents unddsorbed protein, fraction I1 consists of heavy chain. The arrow marks the begin of the elution with NaC1-phosphate buffer, containing 0.5 M 6-aminohexanoic acid
same buffer, containing in addition 0.5 M 6-aminohexanoic acid, pH 7.4, was applied to elute the adsorbed protein, which resulted in the appearance of a second peak in the chromatogram (fraction I1 = heavy chain). The protein-containing fractions were pooled, and the protein was recovered either by precipitation with (NH,),SO, or by ultrafiltration. The (NH,)2S0, precipitation was carried out at pH 7.4 by making the protein solution 2 M in (NH4)2S04.After standing over night at 2 "C the precipitated protein was collected by centrifugation, dissolved in a small amount of appropriate buffer (usually 0.05 M phosphate buffer, 0.1 M NaCl, pH 7.4) and dialyzed against the same buffer medium. For sulfhydryl titration the protein solution was concentrated by ultrafiltration under nitrogen pressure in an Amincon ultrafilter using a UM-10 membrane. During the ultrafiltration process the buffer was gradually replaced by 0.33 M acetate buffer, pH 4.6. The elution of the heavy chain from the adsorbent was also tried with other buffer systems. For this purpose 6-aminohexanoic acid was replaced in the buffer by the same concentration of glycine, p-alanine, Llysine or NaC1. With buffer containing glycine, fi-alanine and NaCl no elution of the adsorbed heavy chain was observed. However, elution occurred with the lysine-containing buffer producing an elution curve practically identical with that obtained with buffer containing 6-aminohexanoic acid. RESULTS Fractionation Procedure
The result of the isolation of the plasmin heavy chain is illustrated in Fig. 2 with polyacrylamide gels of different fractions after dodecylsulfate electrophoresis. Gel A represents plasminogen as the starting
Heavy Chain of Human Plasmin
/il’ P
p’
I
I
I
0
0 OOV0.25
0.50 0.;5 /.60 1.;5 1.50 p-Chloromercuribenzoate added (MI)
Fig. 2. Dodecylsulfhtc-pol).acrylamide electropherogrum qf ( A ) plusminogen, ( B i plasmin, (C) fraction I and (0) fraction II ( = heavy chain). Fractions I and I1 were obtained as indicated in Fig. 1. Migration was from t o p to bottom
Fig. 3. Spectrophotometric titratinn of free su!fhydryl groups of the heayv chain with p-chloromrrcuribenzoare in 0.33 M ucetatc buffer, p H 4.6, containing 6 M urea
material. Gel B shows the protein after 24 h of activation. Due to the reducing medium used in dodecylsulfate electrophoresis the generated plasmin is split intoitslight and heavy chain, thusproducingthecharacteristic two chain pattern. The NH,-terminal peptide moiety released during the activation process [4] is not visible in this system. The two faint bands trailing the heavy chain band may represent traces of protein which was not thoroughly activated. On gel C the protein spectrum of fraction I is shown. This fraction comprises the components which are not adsorbed by polyacrylamide- lysine and which appear in the effluent after the void volume of the column. The electrophoretic pattern indicates that the unadsorbed fraction contains predominantly the plasmin light chain. In addition a minor amount of heavy chain and the two slower moving components which are visible also in gel B can be seen. Fraction I1 is analyzed on gel D. From the comparisonwith the twochain pattern ofplasminit becomes evident that the protein recovered from the polyacrylamide- lysine adsorbent represents essentially pure heavy chain as judged by dodecylsulfate electrophoresis. The electrophoretic mobility corresponds to a component of molecular weight 59000- 61 000. This is identical with the value obtained for the heavy chain fragment in the two chain pattern of plasmin. The comparison between gel C and gel D shows that the plasminlightchainisnot adsorbed bypolyacrylamidelysine and therefore appears in fraction I. The adsorbed and subsequently eluted material of fraction 11 represents practically pure heavy chain and no indication of contaminating light chain was detectable. The plasmin heavy chain was routinely prepared from batches of 60 to 80 mg of plasminogen. The yield of heavy chain was usually between 60 and 65%, as judged from absorbance measurements at 280 nm and from Kjeldahl determinations. After reduction with 2-mercaptoethanol the protein can be carboxymethylated, e . g. with iodoacetic
acid, to block free sulfhydryls. In this case the yield of heavy chain decreases by about 5 to 10 ”/,. The described procedure allows the isolation of the purified heavy chain only. Fraction I which contains the light chain is according to the electrophoretic analyses contaminated with other protein components. For the isolation of pure light chain a different experimental approach or additional purification steps would be necessary. This question is currently under investigation in order to obtain purified light chain for reconstitutional experiments. Titvation of Free Suljlyiryl Groups
When the heavy chain fraction was titrated spectrophotometrically with chloromercuribenzoate in 0.33 M acetate buffer, pH 4.6, an increase in absorbance at 225nm was observed. In the initial titration steps this increase after each addition of chloromercuribenzoate was greater than in later phases of the titration, which when plotted resulted in a graph of two straight lines with different slopes. The intersection point was considered to be the equivalence point. When the titration was carried out in acetate buffer in the absence of urea a plot with an appreciable curvature was obtained. In such cases an exact determination of the equivalence point by graphical extrapolation was difficult. In a series of measurements with different heavy chain preparations in the absence of urea a total of 0.7 - 0.75 mol of free sulfhydryl groups per mol of heavy chain (mol . wt 60000) was obtained. When the titrations were carried out in acetate buffer in the presence of 6 M urea the titration curve followed two straight lines and gave a clear cut equivalence point of 0.9 - 1.O mol of free sulfhydryl groups per mol of heavy chain (Fig. 3). Tn plasminogen and plasmin no free sulfhydryl groups were detectable with the chloromercuribenzoate-method. This result is in agreement with previous findings of Robbins et al. [l].
E. E. Rickli and W . I. Otavsky
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The reduction with 2-mercaptoethanol obviously resulted in the cleavage of a single interchain disulfide bridge, thereby causing the dissociation of the enzyme molecule into its component polypeptide chains. One of the generated free sulfhydryl groups is detectable in the heavy chain portion, whereas the other -SH group must be located in the light chain fragment. Sulfhydryl determinations of the light chain were not carried out, since fraction I in which the light chain appears, is heterogeneous and contains also a significant amount of heavy chain. The above findings confirm previous results of Robbins ~t al. [I] who reported the existence of a single interchain disulfide bridge in human plasmin.
has a value of 3.25 S. The difference in the s-values between the two plateaus is almost 0.9 Svedberg units. Similarly, the diffusion coefficient varies as a function of the 6-aminohexanoic acid concentration (Fig. 5). In contrast to the sedimentation constant the change occurs in the opposite sense. The diffusion coefficient increases between 1 pM and 0.1 M 6-aminohexanoic acid from 4.4 to 5.2 x lo-' cm2 x s-', with a difference in D of 0.8 units. Except for the reciprocal behavior the sedimentation and diffusion curves are very similar in shape.
Ulrracentrijugal Studies The heavy chain sedimented as a single component both in 0.05 M Tris/HCl buffer, pH 7.8, containing 0.1 M NaCl and in the same buffer system containing various concentrations of 6-aminohexanoic acid. Centrifugations at protein concentrations of 2.0, 3.0, 4.5, 6.0 and 7.3 mg/ml revealed that there was practically no concentration dependence of the sedimentation behavior (Fig. 4). Extrapolation of the straight and essentially horizontal line to infinite dilution yielded a sedimentation coelficient of s!o.w = 4.16 S. The sedimentation coefficient at constant protein concentration (4 mg/ml) proved to be highly dependent on thc concentration of 6-aminohexanoic acid in the ~ the 6-aminobuffer medium. The variation of . s , ~ , with hexanoic acid concentration is represented in Fig. 5. At low inhibitor concentrations (1 pM) s approaches asymptotically the value obtained in buffer without inhibitor. In the range between 0.01 and lOmM 6aminohexanoic acid the sedimentation coefficient decreases in a curve of sigmoidal shape and levels off in a lower plateau in the region of lOOmM 6-aminohcxanoic acid. At this inhibitor concentrations s20.w
Fig. 5 . (-)
NH,- Trrniincil Analysis When the heavy chain was subjected to NH,-terminal analysis several terminal amino acid residues were detected by the thin-layer chromatographic technique. In all experiments a dominating spot was observed on the thin-layer sheets which was identified as dansyl threonine. The same result was also obtained with the fluorodinitrobenzene method. Among the other spots which, according to their fluorescence intensities contained only minor amounts of terminal amino acid derivatives, dansyl valine, lysine and glycine could be identified. Previous reports from other laboratories gave different results of the NH,-terminal amino acid composition of the heavy chain of human plasmin. Summaria et a/. [17] originally found NH,-terminal valine, and Wiman and Wallen [4] observed after very short activation times NH,-terminal methionine. The latter authors, however, pointed out that the NH,-terminal region of the heavy chain was apparently quite susceptible to proteolytic degradation under the action of activated plasmin. With increasing activation times this process led to the appearance of NH,-terminal d i n e besides some lysine and methionine. Similar results were obtained in our laboratory. After 1-2 h of activation we also observed as the dominating NH,-
446
terminus of the heavy chain valine and in addition smaller amounts of lysine and methionine. Our present results indicate that the autolytic degradation process proceeds during the 24-h activation period yielding the heavy chain with predominantly NH,-terminal threonine. Enzyme assays showed that the specific plasmin activity of samples activated for 24 h with trace amounts of urokinase in the presence of glycerol was practically identical with the value obtained with the same protein activated according to the modified Remmert-Cohen assay procedure. Thus plasmin with threonine in the NH,-terminal position of its heavy chain is still a potent protease. This species of enzyme molecules may represent a comparatively stable end product within a chain of proteolytic degradation products.
DISCUSSION In its behaviour towards polyacrylamide- lysine the plasmin heavy chain resembles plasminogen which, during its isolation from plasma, is adsorbed specifically by this adsorbent. Also the conditions for the elution of the plasmin heavy chain and of plasminogen from polyacrylamide- lysine appear to be very similar. In the case of plasminogen it is known that buffers containing lysine or chemically related compounds, such as 6-aminohexanoic acid, are suitable for displacing the protein from the adsorbent. The elution experiments carried out with the plasmin heavy chain and with a number of different buffer systems point in the same direction and may indicate an adsorption and elution specificity similar to that observed with plasminogen. The fact that plasminogen as well as plasmin are also adsorbed by polyacrylamide- lysine imposes certain rigid experimental conditions for the isolation of the heavy chain. The first requirement is a complete activation of the proenzyme to plasmin. Secondly, it is necessary to achieve a practically quantitative cleavage of the plasmin into its component polypeptide chains. If these requirements are not fulfilled any residual plasminogen and/or plasmin will also be bound by the adsorbent and will thus show up as impurity in the final heavy chain preparation. The cleavage of the plasmin into its polypeptide chains is accomplished by interchain disulfide reduction. Sulfhydryl titrations of the heavy chain indicated that in the isolated portion only the interchain bridge was cleaved leaving the intrachain disulfides intact. The integrity of the intrachain disulfide bridges seems to be very important for the ability of the heavy chain to adsorb to polyacrylamide- lysine. This property is losr apparently as a consequenceof conformational changes which become possible once the intrachain stabilisation becomes ruptured. For this reason we suspect that the
Heavy Chain of Human Plasmin
amount of heavy chain which does not adsorb to polyacrylamide- lysine and thus becomes lost during the preparation (roughly one third of the theoretical yield) represents material in which the reduction of some intrachain disulfide bridges has occurred. The above assumptions were confirmed in experiments in which the reduction was allowed to proceed beyond 20 min. In these cases a significant decrease of the yield of heavy chain was observed. On the other hand, with a duration of reduction reaction below 20min a band corresponding to the light chain appeared in the dodecylsulfate-electrophoretic pattern (in the presence of reducing agent) indicating that the reductive cleavage of the plasmin was incomplete. The reducing conditions finally chosen give an optimal yield of a practically pure heavy chain preparation. The molecular weight of the heavy chain is in fair agreement with the value published by Wiman and Wallen 1141 and also with results obtained in our laboratory [5] by dodecylsulfate electrophoresis of urokinase-activated, reduced plasmin. The NH,-terminal amino acid determination yielded a result which is not yet described in the literature. According to the apparent susceptibility of the NH,terminal region of the heavy chain to the action of plasmin [4] it must be anticipated that the proteolytic degradation will continue with increasing duration of the activation reaction. It is a surprise, therefore, that under the applied conditions of activation the NH,terminal amino acid pattern is fairly homogeneous, as NH,-terminal threonine clearly dominates. This fact led us to the assumption that the heavy chain undergoes a series of degradation steps which lead to the relatively stable “end product” with NH,-terminal threonine. Fragments which are supposedly liberated during this process must be small since the molecular weight of the heavy chain remained practically constant within the limits of error of the dodecylsulfate electrophoresis. Furthermore, the activity determinations appear to indicate that the degradation process does not involve major alterations of the heavy chain structure. The ultracentrifugal behavior of heavy chain also provides some interesting information. It has been shown by Alkjaersig et al. [18] and later also by other investigators [19,20]that 6-aminohexanoicacid causes a change in the sedimentation coefficient of plasminogen without altering the molecular weight. Analogs of 6-aminohexanoic acid such as lysine and trans-4aminomethyl cyclohexane-1-carboxylic acid have the same effect [19,21]. With their results of rotational diffusion analyses Castellino et nl. [22] reported further evidence for structural changes, since the rotational relaxation time decreased significantly in the presence of 6-aminohexanoic acid and trans-4-aminomethyl cyclohexane-I-carboxylicacid thus signaling the transition to a less rigid protein structure. In gel
E. E. Rickli and W. I. Otavsky
filtration studies Abiko et al. [19] found that 6-aminohexanoic acid and trans-4-aminomethyl cyclohexane1-carboxylic acid bind in a 1: 1 complex to plasminogen indicating a single inhibitor binding site in plasminogen. The fact that the plasmin heavy chain is adsorbed by polyacrylamide - lysine indicates that this plasmin fragment is capable of interacting with 6aminohexanoic acid (and probably also with its analogs). Further evidence for this arises from the ultracentrifugal behavior of the heavy chain. The observed change of s20,was a function of the 6-aminohexanoic acid concentration is very similar to the data reported by Brockway and Castellino [21] on human plasminogen. In both instances this change occurs within the same concentration interval of 6-aminohexanoic acid and also the magnitude of the variation is for both proteins practically the same. Brockway and Castellino [21] determined with increasing amounts of &aminohexanoic acid a decrease of s20,wof close to 1 Svedberg unit for plasminogen, and under similar circumstances s20,wof our heavy chain preparation decreased by about 0.9 Svedberg unit. The conformational alterations reflected by the change of the s-values are further evidenced by a concomitant variation of the diffusion constant. It must be assumed that additional molecular parameters, such as the partial specific volume, will also be affected by 6-aminohexanoic acid. The amount of heavy chain available, however, was not sufficient to allow the determination of the partial specific volume as a function of different concentrations of 6aminohexanoic acid. The reported data indicate that only the heavy chain of human plasmin is capable of combining and interacting with 6-aminohexanoic acid, whereas the light chain which carries the active center [23] is evidently devoid of these properties. It would therefore appear that the inhibition of plasmin by 6-aminohexanoic acid is caused by conformational alterations of the heavy chain alone. The exact nature of the inhibition mechanism, however, is not yet clear. It could be that the conformational changes influence the substrate binding site for which a certain segment of the heavy chain may be of importance. It would also be feasible that the changes of the heavy chain structure induce conformational alterations of the light chain which in turn might affect regions vital for the enzymatic properties.
447 We are indebted to the Central Laboratory of the Blood Transfusion Service of The Swiss Red Cross for the generous supply of plasma. We thank Prof. Dr Hs. Nitschmann of the Institute of Organic Chemistry of the University of Berne for his encouragement and interest in this work. The expert technical assistance of Mrs Verena Kocher is gratefully acknowledged. This investigation was supported by Grant 3.525.71 of the Swiss National Science Foundation.
REFERENCES 1. Robbins, K. C., Summaria, L., Hsieh, B. & Shah, R. J. (1967) J. Biol. Chem. 242, 2333-2342. 2. Walltn, P. & Wiman, B. (1970) Biochim. Biophys. Acia, 221, 20 - 30. 3. Rickli, E. E. & Cuendet, P. A. (1971) Biochim. Biophys. Acta, 250,447-451. 4. Wiman, B. & WallLn, P. (1973) Eur. J . Biochem. 36.25-31. 5. Rickli, E. E. & Otavsky, W. 1. (1973) 4th I n f . Congr. on Thrombosis and Hemostasis, Vienna, Abstr. p. 185, International
Society on Thrombosis and Hemostasis, Vienna. 6. Summaria, L. & Robbins, K. C. (1971) J . Biol. Chem. 246, 2143-2146. 7. Robbins, K. C., Summaria, L., Elwyn, D. & Barlow, G. H. (1965) J . Biol. Chem. 240, 541 - 550. 8. Sanger, F. (1945) Biochem. J . 39, 507-515. 9. Levy, A. L. (1954) Nature (Lond.) 174, 126-127. 10. Brenner, M., Niederwieser, A. & Pataki, G. (1961) Experieniiu (Busel) 17, 145-153. 11. Gros, C. & Labouesse, B. (1969) Eur. J . Biochem. 7,463-470. 12. Gray, W. R. (1972) Methods Enzymol. 25, Part B, 121-138. 13. Woods, K. R. & Wang, K.-T. (1967) Biochim. Biophys. Acta, 133, 369- 370. 14. Crowshaw, K., Jessup, S. J. & Ramwell, P. W. (1967) Biochem. J . 103, 79-85. 15. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244, 44064412. 16. Boyer, P. D. (1954) J . Am. Chem. SOC.76,4331-4337. 17. Summaria. L., Robbins, K. C. & Barlow, G. H. (1971) J . B i d . Chem. 246,2143-2146. 18. Alkjaersig, N., Fletcher, A. P. & Sherry, S. (1959) J . B i d . Chem. 234,832 - 837. 19. Abiko, Y., Iwamota, M. & Tomikawa, M. (1969) Biochim. Biophys. Acta, 185,424-431. 20. Brockway, W. J. & Castellino, F. J . (1971) J . Biol. Chem. 246, 4641 -4647. 21. Brockway, W. J. & Castellino, F. J . (1972) Arch. Biochem. Biophys. 151, 194-199. 22. Castellino, F. J., Brockway, W . J., Thomas, J. K., Liao. H. & Rawitch, A. B. (1973) Biochemistry, 12,2787-2791. 23. Summaria, L., Hsieh, B., Groskopf, W. R., Robbins, K. C. & Barlow, G. H. (1967) J . Biol. Chem. 242, 5046-5052.
E. E. Rickli. Theodor Kocher Institut der Universitlt Bern, Postfach 99, CH-3000 Bern 9. Switzerland W. 1. Otavsky. Department of Biochemistry and Pharmacology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts, U.S.A. 021 1 1
A New Method of Isolation and Some Properties of the Heavy Chain of Human Plasmin Egon E. RICKLI and Wendy I. OTAVSKY Theodor Kocher Institute and Institute of Organic Chemistry, University of Berne (Received October 8, 1974 / August 2. 1975)
A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60- 65 O b ) . Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH,-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
The results from different laboratories have shown that the molecular structures of plasminogen and plasrnin are distinctly different. Plasminogen consists of a single polypeptide chain [ 1 - 31, which is selectively cleaved during the activation process to finally yield the plasmin molecule composed of two polypeptide chains. The two chain structure of plasmin was originally observed by Robbins et al. [l] who reported during activation with urokinase the cleavage of a single arginylvalyl bond in plasminogen, thereby producing a heavy (A) and a light (B) chain which in the native plasmin molecule are apparently connected by a single disulfide bond. The individual chains were demonstrated by starch gel electrophoresis of reduced and carboxymethylated plasmin [l]. The two chain structure of human plasmin was recently confirmed by Wiman and Wallen [4] and also by preliminary results of Rickli and Otavsky [ 5 ] . The isolation and preparation of the individual chains of plasmin first implies the cleavage of the interchain disulfide linkage. Robbins et al. [I] obtained a seemingly preferential cleavage of the interchain disulfide bridge by reduction with 2-mercaptoethanol at Abbreviurion. Dansyl, 5-dimethylaminonaphthalene-1-sulphonyl. Enzyme. Plasmin (EC 3.4.21.7).
low concentration in the presence of Tris buffer, pH 9 in 8 M urea. The cleavage of the intrachain disulfide bonds at alkaline pH apparently requires somewhat stronger reducing conditions. A separation of the two chains of plasmin was achieved by different methods during which extensive reduction and carboxymethylation were usually employed. Essentially pure light and heavy chain preparations were obtained by Wiman and Wallen [4] by gel filtration of the reduced and carboxymethylated plasmin on Sephadex G-150 in 0.1 M Tris, 6 M urea, pH 7.6. Summaria and Robbins [6] prepared purified heavy chain by first dialyzing completely reduced and carboxymethylated plasmin against 0.002 M ammonium bicarbonate thus precipitating most of the light chain. Contaminating amounts of the fragment in the soluble heavy chain fraction were then eliminated by precipitation with 1 M trichloroacetic acid in 8 M urea. In the present study we described a new and simple method for the preparation of the heavy chain of human plasmin. This procedure is based on the ability of the heavy chain fragment, obtained by mild reduction of plasmin, to combine with the adsorbent polyacrylamide-lysine used by us for the isolation of plasminogen from plasma. In addition, we report some properties of the purified heavy chain which, in contrast to other known preparations, has most of its disulfide bonds intact.
Heavy Chain of Human Plasmin
442
MATERIALS AND METHODS Plasminogen
Plasminogen was isolated from 500 - 1000 ml pooled fresh, human ACD-plasma by affinity chromatography as previously published [3]. The elution of the proenzyme from the affinity adsorbent was achieved with 0.3 M phosphate buffer, containing 0.2 M 6-aminohexanoic acid, pH 7.4. Plasminogen was precipitated from the pooled protein-containing fractions with 2 M (NHJ2S04 at pH 7.4, and dialyzed at 2°C for at least 36 h against several changes of 0.05 M Tris-buffer, containing 0.1 M NaC1, pH 7.8 or 9.0. This plasminogen was characterized by glutamic acid as the sole NH,-terminal amino acid residue. The preparations were free of any detectable spontaneous plasmin activity as determined by a caseinolytic assay. The specific activity after activation with streptokinase [3] was between 22 and 27U/mg protein. This is less than previously published [3], and is probably due to larger batches which require a longer time for the completion of the isolation process. Activation of Plasminogen with Urokinase
For the activation of plasminogen the procedure of Robbins et al. [7] was applied in a slightly modified form. Plasminogen was used in a concentration of about 5 mg/ml in 0.05 M Tris-buffer, containing 0.1 M NaCl, pH 7.8, and 25% (v/v) glycerol. Urokinase (generously supplied as “urokinase reagent” [approx. 20 000 units/vial] by Dr R. Strassle, Hoffmann-La Roche, Basel) was then added in a ratio of 1 unit per unit of plasminogen and the activation was allowed to proceed at room temperature for 8 h at which time an additional 0.5 units of urokinase per unit plasminogen was added. Activation was continued for a total of 24 h. NH2-Terminal Amino Acid Analysis
NH,-terminal analyses were carried out with the fluordinitrobenzene method of Sanger [8] in the presence of 8 M urea, and the dinitrophenyl amino acids were analyzed by two-dimensional thin-layer chromatography on silica gel G. In the first dimension the dimension the “toluene solvent system” [9] was used; the solvent of the second dimension was chloroform/ benzyl alcohol/acetic acid (70/30/3, by vol.) [lo]. The results of this method were verified with the dansyl technique. The dansylation procedure of Gros and Labouesse [ l l ] as well as that of Gray [12] was used. After acid hydrolysis at 105- 110 “C for 12- 15 h the samples were dried in V ~ C U Oat 40°C and taken up in 20 - 25 pl of 50 pyridine. For two-dimensional thinlayer chromatography samples of approx. 1 p1 were applied on polyamide sheets (Schleicher & Schull F 1700) coated on both sides. On the reverse side of the
sheets a mixture of standard dansyl amino acids was spotted in the same position as the unknown sample. For the development of the chromatograms the solvent systems of Woods and Wang [13] were used with water/90 % formic acid (200/3, v/v) in the first dimension and benzene/acetic acid (9/1, v/v) in the second dimension. The chromatograms were re-run in the second dimension in ethyl acetate/methanol/acetic acid (20/1/1, by vol.) [14]. Polyacrylamide Electrophoresis
Dodecylsulfate electrophoresis on polyacrylamide gel was camed out according to Weber and Osborn [15]. Gels made from 10% acrylamide and 0.27g of methylene bisacrylamide in 0.1 M phosphate buffer, pH 7.0, containing 0.1 % sodium dodecylsulfate were used. The protein (approx. 0.5 mg/ml) was treated for 2 h at 37 “C with 0.01 M phosphate buffer, pH7.0, containing 1 %, sodium dodecylsulfate and 1 % 2-mercaptoethanol. Samples of 20- 50 pg of protein were applied per gel column (6 x 100mm) and electrophoresis was performed at 8 volts per gel for 6 h after which the gels were stained with Coomassie brilliant blue. For the estimation of molecular weights the following reference proteins were used : human transferrin, bovine glutamate dehydrogenase, hog pepsin, bovine a-chymotrypsinogen A (all purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A.)and human serum albumin (supplied by the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross, Berne). Uliracentrifugal Analyses
Sedimentation and diffusion measurements were carried out in an analytical Spinco, model E, ultracentrifuge at 20 “C. The protein (4 mg/ml) to be analyzed was exhaustively dialyzed at 2 “C against 0.05 M Tris/HCI buffer, pH 7.8, containing 0.1 M NaCl. For one series of measurements this buffer system contained in addition 6-aminohexanoic acid in concentrations varying between 0.1 M and 1 pM. The addition of 6aminohexanoic acid to the buffer always took place before the final pH-adjustment. Sedimentationanalyses were performed in a standard 12 mm cell at 59 780 rev./ min. Diffusion measurements were made in a synthetic boundary cell (valve type, 12 mm) at a speed of 4609 rev./min. Titration of Sulfhydryl Groups
Free sulfhydryl groups were determined according to the spectrophotometric titration method of Boyer [16]. The protein, usually in a concentration of about 4 mg/ml, was titrated in 0.33 M sodium acetate, pH 4.6, containing 8 M urea with p-chloromercuribenzoate
443
E. E. Rickli and W.1. Otdvsky
(Calbiochem) which had been purified by repeated acid precipitations from alkaline solution as recommended by Boyer [16]. Some determinations were also made in the absence of urea. A chloromercuribenzoate solution of approximately 0.6 mM in water (solubilized by a few drops of 0.05 N NaOH) was used. The exact concentration was determined by absorbance measurements at 232 nm at pH 7, using the molar absorption coefficient of 16 900 M-' cm-' as given by Boyer [16]. The titrations were carried out in a Beckman, model DB, spectrophotometer at 225 nm with 3.0 ml of protein solution of known concentration in both the reference and sample cells. To the sample cell were added constant amounts (usually 20 pl) of the chloromercuribenzoate solution. After each addition of titrant the contents of the cell were well mixed and the increase in absorbance was recorded. The absorbance values (corrected for dilution) were then plotted as a function of the chloromercuribenzoate concentration. This resulted in two straight lines with different slopes. The intersection point of the two lines was considered to be the equivalence point of the titration. From the amount of titrant consumed at the equivalence point the amount of free sulfhydryls was calculated.
Preparation and Isolation of the Plasmin Heavy Chain
Plasminogen was activated with urokinase for 24 h as described above. The activated sample was reduced by treatment with 0.1 M 2-mercaptoethanol for 20 min at 20 "C under nitrogen. The mixture was then cooled immediately in ice and applied without delay on a column filled with lysine-substituted polyacrylamide (Biogel P-300), equilibrated in the cold against 0.05 M phosphate buffer, containing 0.1 M NaC1, pH 7.4. The amount of polyacrylamide-lysine was approx. 2 ml of packed gel per mg plasminogen activated. In typical experiments plasminogen in amounts between 60 and 80 mg was used. To insure adequate flow rates acolumn with a cross section of about 20 cm2was chosen. A constant flow rate of 27.2 ml/h was provided with a perspex pump. The column effluent was collected in 5 ml fractions, and the protein content of the fractions was determined from absorbance measurements at 280 nm. The buffers were prepared with boiled water and kept in closed containers under nitrogen. After the protein solution had penetrated into the gel bed the chromatogram was developed by washing the column with 0.05 M phosphate buffer, containing 0.1 M NaCl, pH 7.4. This resulted in the displacement of unadsorbed protein and low molecular weight compounds of the reaction mixture which emerged from the column in a first major peak (fraction I) (Fig. 1). Washing was continued until the absorbance of the effluent had decreased below 0.03. At this point the
Fraction number
Fig. 1. Isolation of the plasmin heavy chain on a column oj'polyacrylamide- lysine. Fraction I represents unddsorbed protein, fraction I1 consists of heavy chain. The arrow marks the begin of the elution with NaC1-phosphate buffer, containing 0.5 M 6-aminohexanoic acid
same buffer, containing in addition 0.5 M 6-aminohexanoic acid, pH 7.4, was applied to elute the adsorbed protein, which resulted in the appearance of a second peak in the chromatogram (fraction I1 = heavy chain). The protein-containing fractions were pooled, and the protein was recovered either by precipitation with (NH,),SO, or by ultrafiltration. The (NH,)2S0, precipitation was carried out at pH 7.4 by making the protein solution 2 M in (NH4)2S04.After standing over night at 2 "C the precipitated protein was collected by centrifugation, dissolved in a small amount of appropriate buffer (usually 0.05 M phosphate buffer, 0.1 M NaCl, pH 7.4) and dialyzed against the same buffer medium. For sulfhydryl titration the protein solution was concentrated by ultrafiltration under nitrogen pressure in an Amincon ultrafilter using a UM-10 membrane. During the ultrafiltration process the buffer was gradually replaced by 0.33 M acetate buffer, pH 4.6. The elution of the heavy chain from the adsorbent was also tried with other buffer systems. For this purpose 6-aminohexanoic acid was replaced in the buffer by the same concentration of glycine, p-alanine, Llysine or NaC1. With buffer containing glycine, fi-alanine and NaCl no elution of the adsorbed heavy chain was observed. However, elution occurred with the lysine-containing buffer producing an elution curve practically identical with that obtained with buffer containing 6-aminohexanoic acid. RESULTS Fractionation Procedure
The result of the isolation of the plasmin heavy chain is illustrated in Fig. 2 with polyacrylamide gels of different fractions after dodecylsulfate electrophoresis. Gel A represents plasminogen as the starting
Heavy Chain of Human Plasmin
/il’ P
p’
I
I
I
0
0 OOV0.25
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Fig. 3. Spectrophotometric titratinn of free su!fhydryl groups of the heayv chain with p-chloromrrcuribenzoare in 0.33 M ucetatc buffer, p H 4.6, containing 6 M urea
material. Gel B shows the protein after 24 h of activation. Due to the reducing medium used in dodecylsulfate electrophoresis the generated plasmin is split intoitslight and heavy chain, thusproducingthecharacteristic two chain pattern. The NH,-terminal peptide moiety released during the activation process [4] is not visible in this system. The two faint bands trailing the heavy chain band may represent traces of protein which was not thoroughly activated. On gel C the protein spectrum of fraction I is shown. This fraction comprises the components which are not adsorbed by polyacrylamide- lysine and which appear in the effluent after the void volume of the column. The electrophoretic pattern indicates that the unadsorbed fraction contains predominantly the plasmin light chain. In addition a minor amount of heavy chain and the two slower moving components which are visible also in gel B can be seen. Fraction I1 is analyzed on gel D. From the comparisonwith the twochain pattern ofplasminit becomes evident that the protein recovered from the polyacrylamide- lysine adsorbent represents essentially pure heavy chain as judged by dodecylsulfate electrophoresis. The electrophoretic mobility corresponds to a component of molecular weight 59000- 61 000. This is identical with the value obtained for the heavy chain fragment in the two chain pattern of plasmin. The comparison between gel C and gel D shows that the plasminlightchainisnot adsorbed bypolyacrylamidelysine and therefore appears in fraction I. The adsorbed and subsequently eluted material of fraction 11 represents practically pure heavy chain and no indication of contaminating light chain was detectable. The plasmin heavy chain was routinely prepared from batches of 60 to 80 mg of plasminogen. The yield of heavy chain was usually between 60 and 65%, as judged from absorbance measurements at 280 nm and from Kjeldahl determinations. After reduction with 2-mercaptoethanol the protein can be carboxymethylated, e . g. with iodoacetic
acid, to block free sulfhydryls. In this case the yield of heavy chain decreases by about 5 to 10 ”/,. The described procedure allows the isolation of the purified heavy chain only. Fraction I which contains the light chain is according to the electrophoretic analyses contaminated with other protein components. For the isolation of pure light chain a different experimental approach or additional purification steps would be necessary. This question is currently under investigation in order to obtain purified light chain for reconstitutional experiments. Titvation of Free Suljlyiryl Groups
When the heavy chain fraction was titrated spectrophotometrically with chloromercuribenzoate in 0.33 M acetate buffer, pH 4.6, an increase in absorbance at 225nm was observed. In the initial titration steps this increase after each addition of chloromercuribenzoate was greater than in later phases of the titration, which when plotted resulted in a graph of two straight lines with different slopes. The intersection point was considered to be the equivalence point. When the titration was carried out in acetate buffer in the absence of urea a plot with an appreciable curvature was obtained. In such cases an exact determination of the equivalence point by graphical extrapolation was difficult. In a series of measurements with different heavy chain preparations in the absence of urea a total of 0.7 - 0.75 mol of free sulfhydryl groups per mol of heavy chain (mol . wt 60000) was obtained. When the titrations were carried out in acetate buffer in the presence of 6 M urea the titration curve followed two straight lines and gave a clear cut equivalence point of 0.9 - 1.O mol of free sulfhydryl groups per mol of heavy chain (Fig. 3). Tn plasminogen and plasmin no free sulfhydryl groups were detectable with the chloromercuribenzoate-method. This result is in agreement with previous findings of Robbins et al. [l].
E. E. Rickli and W . I. Otavsky
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Ejyeect of' 6-ominohe.xanoic ocid on /he .redimenraticm and diffusion (A-A) consfants of the plusmin heavy chain. The negative logarithm of the 6-aminohexanoic acid concentration is plotted
The reduction with 2-mercaptoethanol obviously resulted in the cleavage of a single interchain disulfide bridge, thereby causing the dissociation of the enzyme molecule into its component polypeptide chains. One of the generated free sulfhydryl groups is detectable in the heavy chain portion, whereas the other -SH group must be located in the light chain fragment. Sulfhydryl determinations of the light chain were not carried out, since fraction I in which the light chain appears, is heterogeneous and contains also a significant amount of heavy chain. The above findings confirm previous results of Robbins ~t al. [I] who reported the existence of a single interchain disulfide bridge in human plasmin.
has a value of 3.25 S. The difference in the s-values between the two plateaus is almost 0.9 Svedberg units. Similarly, the diffusion coefficient varies as a function of the 6-aminohexanoic acid concentration (Fig. 5). In contrast to the sedimentation constant the change occurs in the opposite sense. The diffusion coefficient increases between 1 pM and 0.1 M 6-aminohexanoic acid from 4.4 to 5.2 x lo-' cm2 x s-', with a difference in D of 0.8 units. Except for the reciprocal behavior the sedimentation and diffusion curves are very similar in shape.
Ulrracentrijugal Studies The heavy chain sedimented as a single component both in 0.05 M Tris/HCl buffer, pH 7.8, containing 0.1 M NaCl and in the same buffer system containing various concentrations of 6-aminohexanoic acid. Centrifugations at protein concentrations of 2.0, 3.0, 4.5, 6.0 and 7.3 mg/ml revealed that there was practically no concentration dependence of the sedimentation behavior (Fig. 4). Extrapolation of the straight and essentially horizontal line to infinite dilution yielded a sedimentation coelficient of s!o.w = 4.16 S. The sedimentation coefficient at constant protein concentration (4 mg/ml) proved to be highly dependent on thc concentration of 6-aminohexanoic acid in the ~ the 6-aminobuffer medium. The variation of . s , ~ , with hexanoic acid concentration is represented in Fig. 5. At low inhibitor concentrations (1 pM) s approaches asymptotically the value obtained in buffer without inhibitor. In the range between 0.01 and lOmM 6aminohexanoic acid the sedimentation coefficient decreases in a curve of sigmoidal shape and levels off in a lower plateau in the region of lOOmM 6-aminohcxanoic acid. At this inhibitor concentrations s20.w
Fig. 5 . (-)
NH,- Trrniincil Analysis When the heavy chain was subjected to NH,-terminal analysis several terminal amino acid residues were detected by the thin-layer chromatographic technique. In all experiments a dominating spot was observed on the thin-layer sheets which was identified as dansyl threonine. The same result was also obtained with the fluorodinitrobenzene method. Among the other spots which, according to their fluorescence intensities contained only minor amounts of terminal amino acid derivatives, dansyl valine, lysine and glycine could be identified. Previous reports from other laboratories gave different results of the NH,-terminal amino acid composition of the heavy chain of human plasmin. Summaria et a/. [17] originally found NH,-terminal valine, and Wiman and Wallen [4] observed after very short activation times NH,-terminal methionine. The latter authors, however, pointed out that the NH,-terminal region of the heavy chain was apparently quite susceptible to proteolytic degradation under the action of activated plasmin. With increasing activation times this process led to the appearance of NH,-terminal d i n e besides some lysine and methionine. Similar results were obtained in our laboratory. After 1-2 h of activation we also observed as the dominating NH,-
446
terminus of the heavy chain valine and in addition smaller amounts of lysine and methionine. Our present results indicate that the autolytic degradation process proceeds during the 24-h activation period yielding the heavy chain with predominantly NH,-terminal threonine. Enzyme assays showed that the specific plasmin activity of samples activated for 24 h with trace amounts of urokinase in the presence of glycerol was practically identical with the value obtained with the same protein activated according to the modified Remmert-Cohen assay procedure. Thus plasmin with threonine in the NH,-terminal position of its heavy chain is still a potent protease. This species of enzyme molecules may represent a comparatively stable end product within a chain of proteolytic degradation products.
DISCUSSION In its behaviour towards polyacrylamide- lysine the plasmin heavy chain resembles plasminogen which, during its isolation from plasma, is adsorbed specifically by this adsorbent. Also the conditions for the elution of the plasmin heavy chain and of plasminogen from polyacrylamide- lysine appear to be very similar. In the case of plasminogen it is known that buffers containing lysine or chemically related compounds, such as 6-aminohexanoic acid, are suitable for displacing the protein from the adsorbent. The elution experiments carried out with the plasmin heavy chain and with a number of different buffer systems point in the same direction and may indicate an adsorption and elution specificity similar to that observed with plasminogen. The fact that plasminogen as well as plasmin are also adsorbed by polyacrylamide- lysine imposes certain rigid experimental conditions for the isolation of the heavy chain. The first requirement is a complete activation of the proenzyme to plasmin. Secondly, it is necessary to achieve a practically quantitative cleavage of the plasmin into its component polypeptide chains. If these requirements are not fulfilled any residual plasminogen and/or plasmin will also be bound by the adsorbent and will thus show up as impurity in the final heavy chain preparation. The cleavage of the plasmin into its polypeptide chains is accomplished by interchain disulfide reduction. Sulfhydryl titrations of the heavy chain indicated that in the isolated portion only the interchain bridge was cleaved leaving the intrachain disulfides intact. The integrity of the intrachain disulfide bridges seems to be very important for the ability of the heavy chain to adsorb to polyacrylamide- lysine. This property is losr apparently as a consequenceof conformational changes which become possible once the intrachain stabilisation becomes ruptured. For this reason we suspect that the
Heavy Chain of Human Plasmin
amount of heavy chain which does not adsorb to polyacrylamide- lysine and thus becomes lost during the preparation (roughly one third of the theoretical yield) represents material in which the reduction of some intrachain disulfide bridges has occurred. The above assumptions were confirmed in experiments in which the reduction was allowed to proceed beyond 20 min. In these cases a significant decrease of the yield of heavy chain was observed. On the other hand, with a duration of reduction reaction below 20min a band corresponding to the light chain appeared in the dodecylsulfate-electrophoretic pattern (in the presence of reducing agent) indicating that the reductive cleavage of the plasmin was incomplete. The reducing conditions finally chosen give an optimal yield of a practically pure heavy chain preparation. The molecular weight of the heavy chain is in fair agreement with the value published by Wiman and Wallen 1141 and also with results obtained in our laboratory [5] by dodecylsulfate electrophoresis of urokinase-activated, reduced plasmin. The NH,-terminal amino acid determination yielded a result which is not yet described in the literature. According to the apparent susceptibility of the NH,terminal region of the heavy chain to the action of plasmin [4] it must be anticipated that the proteolytic degradation will continue with increasing duration of the activation reaction. It is a surprise, therefore, that under the applied conditions of activation the NH,terminal amino acid pattern is fairly homogeneous, as NH,-terminal threonine clearly dominates. This fact led us to the assumption that the heavy chain undergoes a series of degradation steps which lead to the relatively stable “end product” with NH,-terminal threonine. Fragments which are supposedly liberated during this process must be small since the molecular weight of the heavy chain remained practically constant within the limits of error of the dodecylsulfate electrophoresis. Furthermore, the activity determinations appear to indicate that the degradation process does not involve major alterations of the heavy chain structure. The ultracentrifugal behavior of heavy chain also provides some interesting information. It has been shown by Alkjaersig et al. [18] and later also by other investigators [19,20]that 6-aminohexanoicacid causes a change in the sedimentation coefficient of plasminogen without altering the molecular weight. Analogs of 6-aminohexanoic acid such as lysine and trans-4aminomethyl cyclohexane-1-carboxylic acid have the same effect [19,21]. With their results of rotational diffusion analyses Castellino et nl. [22] reported further evidence for structural changes, since the rotational relaxation time decreased significantly in the presence of 6-aminohexanoic acid and trans-4-aminomethyl cyclohexane-I-carboxylicacid thus signaling the transition to a less rigid protein structure. In gel
E. E. Rickli and W. I. Otavsky
filtration studies Abiko et al. [19] found that 6-aminohexanoic acid and trans-4-aminomethyl cyclohexane1-carboxylic acid bind in a 1: 1 complex to plasminogen indicating a single inhibitor binding site in plasminogen. The fact that the plasmin heavy chain is adsorbed by polyacrylamide - lysine indicates that this plasmin fragment is capable of interacting with 6aminohexanoic acid (and probably also with its analogs). Further evidence for this arises from the ultracentrifugal behavior of the heavy chain. The observed change of s20,was a function of the 6-aminohexanoic acid concentration is very similar to the data reported by Brockway and Castellino [21] on human plasminogen. In both instances this change occurs within the same concentration interval of 6-aminohexanoic acid and also the magnitude of the variation is for both proteins practically the same. Brockway and Castellino [21] determined with increasing amounts of &aminohexanoic acid a decrease of s20,wof close to 1 Svedberg unit for plasminogen, and under similar circumstances s20,wof our heavy chain preparation decreased by about 0.9 Svedberg unit. The conformational alterations reflected by the change of the s-values are further evidenced by a concomitant variation of the diffusion constant. It must be assumed that additional molecular parameters, such as the partial specific volume, will also be affected by 6-aminohexanoic acid. The amount of heavy chain available, however, was not sufficient to allow the determination of the partial specific volume as a function of different concentrations of 6aminohexanoic acid. The reported data indicate that only the heavy chain of human plasmin is capable of combining and interacting with 6-aminohexanoic acid, whereas the light chain which carries the active center [23] is evidently devoid of these properties. It would therefore appear that the inhibition of plasmin by 6-aminohexanoic acid is caused by conformational alterations of the heavy chain alone. The exact nature of the inhibition mechanism, however, is not yet clear. It could be that the conformational changes influence the substrate binding site for which a certain segment of the heavy chain may be of importance. It would also be feasible that the changes of the heavy chain structure induce conformational alterations of the light chain which in turn might affect regions vital for the enzymatic properties.
447 We are indebted to the Central Laboratory of the Blood Transfusion Service of The Swiss Red Cross for the generous supply of plasma. We thank Prof. Dr Hs. Nitschmann of the Institute of Organic Chemistry of the University of Berne for his encouragement and interest in this work. The expert technical assistance of Mrs Verena Kocher is gratefully acknowledged. This investigation was supported by Grant 3.525.71 of the Swiss National Science Foundation.
REFERENCES 1. Robbins, K. C., Summaria, L., Hsieh, B. & Shah, R. J. (1967) J. Biol. Chem. 242, 2333-2342. 2. Walltn, P. & Wiman, B. (1970) Biochim. Biophys. Acia, 221, 20 - 30. 3. Rickli, E. E. & Cuendet, P. A. (1971) Biochim. Biophys. Acta, 250,447-451. 4. Wiman, B. & WallLn, P. (1973) Eur. J . Biochem. 36.25-31. 5. Rickli, E. E. & Otavsky, W. 1. (1973) 4th I n f . Congr. on Thrombosis and Hemostasis, Vienna, Abstr. p. 185, International
Society on Thrombosis and Hemostasis, Vienna. 6. Summaria, L. & Robbins, K. C. (1971) J . Biol. Chem. 246, 2143-2146. 7. Robbins, K. C., Summaria, L., Elwyn, D. & Barlow, G. H. (1965) J . Biol. Chem. 240, 541 - 550. 8. Sanger, F. (1945) Biochem. J . 39, 507-515. 9. Levy, A. L. (1954) Nature (Lond.) 174, 126-127. 10. Brenner, M., Niederwieser, A. & Pataki, G. (1961) Experieniiu (Busel) 17, 145-153. 11. Gros, C. & Labouesse, B. (1969) Eur. J . Biochem. 7,463-470. 12. Gray, W. R. (1972) Methods Enzymol. 25, Part B, 121-138. 13. Woods, K. R. & Wang, K.-T. (1967) Biochim. Biophys. Acta, 133, 369- 370. 14. Crowshaw, K., Jessup, S. J. & Ramwell, P. W. (1967) Biochem. J . 103, 79-85. 15. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244, 44064412. 16. Boyer, P. D. (1954) J . Am. Chem. SOC.76,4331-4337. 17. Summaria. L., Robbins, K. C. & Barlow, G. H. (1971) J . B i d . Chem. 246,2143-2146. 18. Alkjaersig, N., Fletcher, A. P. & Sherry, S. (1959) J . B i d . Chem. 234,832 - 837. 19. Abiko, Y., Iwamota, M. & Tomikawa, M. (1969) Biochim. Biophys. Acta, 185,424-431. 20. Brockway, W. J. & Castellino, F. J . (1971) J . Biol. Chem. 246, 4641 -4647. 21. Brockway, W. J. & Castellino, F. J . (1972) Arch. Biochem. Biophys. 151, 194-199. 22. Castellino, F. J., Brockway, W . J., Thomas, J. K., Liao. H. & Rawitch, A. B. (1973) Biochemistry, 12,2787-2791. 23. Summaria, L., Hsieh, B., Groskopf, W. R., Robbins, K. C. & Barlow, G. H. (1967) J . Biol. Chem. 242, 5046-5052.
E. E. Rickli. Theodor Kocher Institut der Universitlt Bern, Postfach 99, CH-3000 Bern 9. Switzerland W. 1. Otavsky. Department of Biochemistry and Pharmacology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts, U.S.A. 021 1 1
