- Email: [email protected]
A single-step separation of the one- and two-chain forms of tissue plasminogen activator
ARCHIVES
Vol.
OF BIOCHEMISTRY
285, No. 2, March,
AND
BIOPHYSICS
pp. 373-376,
1991
COMMUNICATION A Single-Step Separation of the One- and Two-Chain Forms of Tissue Plasminogen Activator’ S. Shaukat Department
Received
Husain of
August
Anesthesia,
Massachusetts
24, 1990, and in revised
form
General Hospital
November
and Harvard
Medical
Tissue plasminogen activator (tPA)’ is synthesized and secreted as a one-chain glycoprotein (1). Most tPA-producing cells secrete a mixture of 63- and 65kDa forms, which differ from
one another in the number of sites that are glycosylated. The 65-kDa variant of tPA is glycosylated at Asn-117 of the kringle 1 domain, Asn-184 of the kringle 2 domain, and Asn-448 of the protease domain, while the 63-kDa form is glycosylated at Asn residues 117 and 448 only (2). Cleavage of the peptide bond between Arg-275 and Ile-276 of one-chain tPA by plasmin or some other trypsin-like serine proteases generates two-chain tPA, consisting of two-polypeptide chains, linked by a single disulfide bond (3). The mature secreted form of one-chain tPA, unlike one-chain zymogen forms of most other serine proteases, which show essentially no enzymatic activity, possesses considerable activity (4). However, significant differences do exist between one-chain and two-chain forms of tPA in their enzymatic and other physicochemical properties. Elucidation of these differences has been a focus of much attention to understand the physiological significance for the conversion of one-chain to two-chain tPA (5). Purification of tPA in the absence of a protease inhibitor, such as aprotinin, results in the cleavage of one-chain tPA by endogenous proteases, yielding primarily the two-chain form tPA (6). If aprotinin is present during purification, proteolytic cleavage of one-chain tPA is inhibited and the predominant product is one-chain tPA (6, 7). However, most purified preparations of one-chain tPA are contaminated with variable amounts of two-chain tPA. There is no effective procedure currently available for the removal of two-chain tPA contaminant from one-chain tPA preparations. Consequently, much of the work on enzymatic properties of one-chain tPA has been carried out with preparations contaminated with variable amounts of two-chain tPA. Here 1 describe a simple and effective procedure of affinity chromatography on benzamidine-Sepharose that removes two-chain contaminant and concomitantly separates differentially glycosylated one-chain variants from preparations of one-chain tPA. Some of the data described in this paper have been presented at the Tenth International Congress on Fibrinolysis.
EXPERIMENTAL ‘This work was supported by Research Grant HL38178 from the National Institutes of Health. ’ Abbreviations used: tPA, tissue plasminogen activator; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin. $3.00 0 1991 by Academic Press, of reproduction in any form
02114
29, 1990
Preparations of one-chain tissue plasminogen activator @PA), usually a mixture of 65- and 63-kDa differentially glycosylated forms, contain variable amounts of twochain tPA. There is no effective procedure currently available for removal of the two-chain contaminant from one-chain tPA preparations. In this report, affinity chromatography on benzamidine-Sepharose was investigated for the separation of two-chain from one-chain tPA. Activase, a preparation of recombinant tPA containing 80% one-chain tPA, a mixture of 65- and 63-kDa variants, and 20% two-chain tPA, was applied to a column of benzamidine-Sepharose, equilibrated with 1 M ammonium bicarbonate. Under this condition, both one-chain and two-chain forms of tPA were adsorbed by the column. Addition of 0.1 M arginine to the equilibration buffer led to elution of two-peaks, corresponding to the 65- and 63kDa variants of one-chain tPA. Two-chain tPA remained bound to the column, but could be eluted with sodium acetate buffer, pH 4.0, containing 0.1 M arginine. The present procedure allows rapid and effective removal of two-chain tPA with concomitant separation of 65- and 63-kDa one-chain glycoforms from preparations of onechain tPA. Kinetic analysis for the hydrolysis of D-IkPro-Arg-p-nitroanilide (S-2288) by the highly purified molecular forms of tPA suggests that 63-kDa one-chain tPA possesses 30% higher catalytic efficiency than the 65-kDa variant, while two-chain tPA is 9- or la-fold more efficient than 63- or 65-kDa one-chain tPA, respectively. 0 1991 Academic Press, Inc.
0003-9861/91 Copyright All rights
School, Boston, Massachusetts
PROCEDURES
Material. Activase (Alteplase), a preparation of recombinant tPA, was produced by Genentech (San Francisco, CA). The preparation contained 80% one-chain tPA, a mixture of 65- and 63-kDa forms, and 20% two-chain tPA. A preparation of single-chain tPA, containing 90-95% 373
Inc. reserved.
374
S. SHAUKAT
Fraction
HUSAIN
Number
FIG. 1. Chromatography of Activase (a preparation of one-chain recombinant tPA) on a benzamidine-Sepharose column. The tPA preparation in 1 M ammonium bicarbonate was chromatographed on the column as described in the Results section. Fractions (1 ml) were collected at 7 ml/ h. After the column was washed with the equilibration buffer, elution was performed with 0.1 M arginine in 1 M ammonium bicarbonate at the arrow marked “a” and with 0.1 M sodium acetate, 1 M NaCl, 0.3 M arginine, pH 4, at the arrow marked “b”. single-chain form, derived from mammalian cell culture was obtained from Calbiochem. Chromogenic substrate for tPA, D-Ile-L-Pro-L-Argp-nitroanilide (S-2288), was obtained from Kabi Vitrum, Stockholm, Sweden. Prepacked gel filtration columns, Econo-Pat lODG, were obtained from Bio-Rad, (Richmond, CA). Centricon 10 microconcentrator was obtained from Amicon (Danvers, MA). Other chemicals were obtained from the following sources: 6-aminohexanoic acid coupled to Sepharose 4B (CH Sepharose 4B) and benzamidine-agarose @-aminobenzamidine coupled to agarose via the spacer glycyl-glycine), Sigma; N-ethyl-N-(3dimethylaminopropyl)carbodiimide hydrochloride and 4aminobenzamidine hydrochloride, Aldrich, protein grade Tween 80, Calbiochem. Methods. Benzamidine-Sepharose was prepared by coupling 4-aminobenzamidine to CL-Sepharose in the presence of iV-ethyl-iV’-(3methylaminopropyl)carbodiimide as described by Holmberg et al. (8). The derivative was extensively washed to remove by-products of the reaction as described in the procedure. Polyacrylamide SDS gel electrophoresis of tPA samples, reduced with 20 mM dithiothreitol for 3 min at lOO’C, was performed on 10% slab gels by the method of Laemmli (9). The gels were stained with Coomassie brilliant blue. Amidolytic activity of tPA was determined with 0.5 mM S-2288 in 0.05 M Tris-HCl, 0.15 M NaCl, 20 KIU/ml Trasylol, 1 mg/ml BSA, 0.01% Tween 80, pH 7.4, at 37’C. The increase in absorbance at 406 nm was continuously monitored using a Hewlett-Packard diode array spectrophotometer. For kinetic analysis of the amidolytic activities of various molecular forms of tPA, assays were performed with the substrate concentration varying from 0.0625 to 2 mM. Kinetic parameters were determined from Eadie-Hofstee (10) and Lineweaver-Burk (11) plots using linear regression analyses. RESULTS
Enzyme Purification Activase (50 mg vial, containing 1.7 g arginine, 0.5 g phosphate, and 4 mg polysorbate 80) was reconstituted with 25 ml water. To remove arginine and other salts, an aliquot of the Activase solution (2.5 ml containing 5 mg protein) was passed through Econo-Pat 1ODG column (10 ml bed volume) equilibrated with 1 M ammonium bicarbonate (adjusted to pH 8.0). The protein emerged as a sharp peak in the breakthrough fractions.
The pooled, gel-filtered tPA solution (7.6 ml containing 8.5 mg protein) from two Econo-Pat 1ODG columns was applied at 7 ml/h to a column of benzamidine-Sepharose (1 X 20 cm, 10 ml volume) equilibrated with 1 M ammonium bicarbonate (adjusted to pH 8.0). After washing with one column volume of the equilibrium buffer, elution was started with 1 M ammonium bicarbonate containing 0.1 M arginine (adjusted to pH 8.0). This resulted in the elution of two peaks with activities toward S2288 and plasminogen. Further washing of the column with 0.1 M sodium acetate, 1 M NaCl, 0.3 M arginine, pH 4.0, led to the elution of a third enzymatically active peak (Fig. 1). Fractions 34-46 (Peak l), 57-75 (Peak 2), 47-56 (region between Peaks 1 and 2), and 82-88 (Peak 3) were concentrated in a Centricon 10 concentrator to give solutions with protein concentrations of approximately 1 mg/ml. SDS-polyacrylamide gel electrophoresis of the starting tPA showed that the preparation was a mixture of 80% one-chain and 20% two-chain tPA. The one-chain tPA band comprised two closely spaced 65- and 63-kDa bands (not clearly distinguishable because of overloading in the gel shown). The first peak eluting with 0.1 M arginine-ammonium bicarbonate from the benzamidine-Sepharose column contained 65-kDA onechain tPA, while the closely following second peak contained 63-kDa one-chain tPA. The area between the two peaks was mostly 63-kDa tPA with some 65-kDa variant. The tPA peak eluting with the acetate buffer contained essentially pure twochain tPA (Fig. 2). The purification procedure was repeated with a different preparation of single-chain tPA (Calbiochem) as well as with a commercially available preparation of immobilized benzamidine that has the ligand attached to agarose via glycyl-glycine spacer (Sigma). Chromatography of the single-chain tPA preparation on benzamidine immobilized on Sepharose via aminohexanoic acid spacer resulted in the separation of single-chain and twochain forms of tPA as described for Activase. Chromatography of the tPA preparation on benzamidine attached to agarose via glycyl-glycine resulted in the adsorption of various forms of tPA by the affinity matrix. However, the bound tPA slowly leeched
SEPARATION
OF
ONE-
AND
TWO-CHAIN
FIG. 2. SDS gel electrophoresis of reduced tPA fractions: Protein bands were stained with Coomassie blue. The molecular marker contained phosphorylase (92 kDa), BSA (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa). Lane 1, molecular-weight markers; Lane 2, starting t PA; Lane 3, fractions 34-46 (Peak l), 65-kDa one-chain t PA, Lane 4, fractions 47-56 (region between Peak 1 and 2); Lane 5, fractions 5775 (Peak 2), 63-kDa one-chain tPA; Lane 6, fractions 82-88 (Peak 3), two-chain t PA; Lane 7, molecular-weight markers.
out with the equilibration buffer and application of the argininecontaining buffer caused elution of various forms of tPA in one peak. This suggests that the benzamidine immobilized via the glycyl-glycine spacer binds tPA less tightly than the ligand immobilized through the aminohexanoic acid arm.
Kinetics of Hydrolysis of S-2288 by the Purified TwoChain and 65- and 63-kDa One-Chain Forms of tPA Kinetic constants for the hydrolysis of S-2288 by two-chain tPA and the 65- and 63-kDa variants of one-chain tPA are given in Table I. The values determined from Lineweaver-Burk or Eadie-Hofstee plots are in close agreement. Hydrolysis of the chromogenic substrate by two-chain tPA is characterized by lower K,,, and higher K,,, values than those of the one-chain variants. The resulting catalytic efficiency of two-chain tPA for S-2288 is 12 and 9 times higher than those of the 65- and 63kDa one-chain forms of tPA, respectively. Both K,,, and k,, values of 65-kDa one-chain tPA are higher than the corresponding values of the 63-kDa one-chain variant. The difference in the K,,, is comparatively more than the difference in the k,, values. The catalytic efficiency of 63-kDa one-chain tPA is therefore about 30% higher than that of the 65 kDa variant.
TISSUE
PLASMINOGEN
one-chain tPA. The procedure also results in the separation of 65- and 63-kDa variants of one-chain tPA. The differentially glycosylated variants of tPA have been previously separated by gradient elution from arginineSepharose (12) or lysine-Sepharose (13-15). However, these procedures fail to separate two-chain tPA from onechain tPA. The present method provides a rapid one-step procedure for the removal of two-chain tPA with concomitant separation of one-chain tPA variants. The method provides a comparatively inexpensive source of highly purified 65- and 63-kDa variants of one-chain tPA and the by-product two-chain tPA from clinical preparations. tPA undergoes extensive aggregation and precipitation at concentrations higher than 50 pg per milliliter, except in the presence of chaotropic agents, such as arginine, ammonium thiocyanate, or ammonium bicarbonate that inhibit aggregation. The effectiveness of the benzamidineSepharose column in 1 M ammonium bicarbonate greatly facilitated the present separation procedure. The kinetic efficiencies of 65- and 63-kDa one-chain tPA for S-2288 were found to be 8 and lo%, respectively, of that of two-chain tPA. Earlier figures obtained with mixtures of one-chain 65- and 63-kDa variants contaminated with varying quantities of two-chain tPA suggested catalytic efficiencies of 36% (16), 26% (17), and 17% (18) for one-chain tPA compared to that of two-chain tPA. The present values indicating a larger difference between the activities of one-chain and two-chain tPA reflect a higher degree of enzyme purity. The present investigation also suggests that glycosylation at the Asn-117 residue for the hydrolysis S-2288 affects the kinetic parameters by one-chain tPA. The catalytic efficiency of the 63-kDa one-chain variant, lacking the carbohydrate moiety at Asn-117, is slightly higher than that of the 65-kDa variant. Kinetic parameters for the amidolytic activities of the 63and 65-kDa variants of one-chain tPA have not been previously determined. However, a recent investigation (19) suggests that two-chain tPA unglycosylated at Asn-117 TABLE
tPA is enzymatically active, chroSince one-chain matographic procedures or inhibitor treatment that are usually used to remove active two-chain from inactive one-chain forms of serine proteases are believed to be ineffective for the removal of two-chain tPA from preparations of one-chain tPA. However, this report shows that chromatography of tPA on benzamidine-Sepharose results in an effective separation of two-chain tPA from
I
Kinetic Constants for the Hydrolysis of D-Ile-Pro-Arg-pNitroanilide (S-2288) by Various Molecular Forms of tPA Form
DISCUSSION
375
ACTIVATOR
of tPA
K,,, (mM)
kc., W')
kcat/Km 6.24 + 0.18 (6.4 _+ 0.24)
65-kDa
one-chain
tPA
2.0 (1.8
f 0.1 f 0.1)
12.3 + 0.24 (11.5 +- 0.58)
63-kDa
one-chain
tPA
1.1 + 0.1 (0.93 + 0.1)
8.7 f 0.55 (7.7 * 0.51)
7.7 (8.3
rl: 0.22 + 0.39)
0.40 k 0.02 (0.37 f 0.02)
30.2 zk 0.15 (29.0 z!z 0.15)
76.1 (78.2
+- 3.13 +- 4.1)
Two-chain
tPA
Note. Data are the means of three experiments + SD. The kinetic parameters were calculated from Eadie-Hofstee (10) plot and Lineweaver-Burk (11) plot (values in parentheses).
376
S. SHAUKAT
has higher plasminogen activator activity than the variant with a carbohydrate chain at this residue.
HUSAIN
8.
Holmberg, L., Bladh, Acta 445,215-222.
9. Laemmli,
ACKNOWLEDGMENT The author in performing
Zaidi
for technical
assistance
Lett.
2. Pohl, G., Kallistrom,
M., Bergsdorf, N., Wallen, H. (1984) Biochemistry 23,3701-3707. Ichinose, A., Kisiel, W., and Fujikawa, K. (1984) 412-418.
P., and Jornvall, FEBS
I&t.
1’75,
4. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257,2920-2925. 5. Husain, S. S., Hasan, A. K. H., and Budzynski, A. Z. (1989) Blood 74, 999-1006. 6. Rijken, D. C., and Collen, D. (1981) J. Biol. Chem. 256,7035-7041. 7. Wallen, P., Ranby, M., Bergsdorf, N., and Kok, P. (1981) in Progress in Fibrinolysis (Davidson, Vol. 5, pp. 16-23, Churchill
J. F., Nilsson, Livingstone,
I. M., Astedt, Edinburgh.
B., Eds.),
H., and Burk, B. H. J. (1952)
12. Ranby,
1. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., and Collen, D. (1983) Nature (London) 301, 214-221
Nature
B. (1976)
(London)
D. (1934)
Biochem.
Biophys.
227.680-685.
J. Amer.
Chem.
Sot. 56,658-
666. 11. Hofstee,
REFERENCES
3.
U. K. (1970)
10. Lineweaver,
thanks Mr. Syed Tanweer gel electrophoreses.
B., and Astedt,
M.,
Bergsdorf,
Science N., Pohl,
116, 329-331. G., and Wallen,
13. Einarsson, M., Brandt, J., and Kaplan, L. Actu (1985) 830, l-10. 14 Parekh, R. B., Dwek, R. A., Thomas, J. demacher, T. W., Wittwer, A. J., Howard, N. R., Jennings, M. G., Harakas, N. K., ch.emis0-y 28,7644-7662. 15 Wittwer, A. J., and Howard, S. C. (1990) 4180.
16. Urano, Arch.
P. (1982)
FEBS
146,289-292. (1985)
Biochim.
R., Opendakker, G., RaS. C., Nelson, R., Siegel, and Feder, J. (1989) BioBiochemistry
T., deserrano, V. S., Urano, S., and Castellino, Biochem. Biophys. 270, 356-362.
17. Loscalzo,
J. (1988)
18. Ranby, M., Bergsdorf, 175-183.
J. Clin. Inuest.
Biophys.
82,
1391-1397.
N., and Nilsson,
T. (1982)
29,
F. J., (1989)
Thromb.
19. Wittwer, A. J., Howard, S. C., Carr, L. S., Harakas, Feder, J. (1989) Biochemistry 28, 7662-7669.
4175-
Res. 27, N. K., and
Vol.
OF BIOCHEMISTRY
285, No. 2, March,
AND
BIOPHYSICS
pp. 373-376,
1991
COMMUNICATION A Single-Step Separation of the One- and Two-Chain Forms of Tissue Plasminogen Activator’ S. Shaukat Department
Received
Husain of
August
Anesthesia,
Massachusetts
24, 1990, and in revised
form
General Hospital
November
and Harvard
Medical
Tissue plasminogen activator (tPA)’ is synthesized and secreted as a one-chain glycoprotein (1). Most tPA-producing cells secrete a mixture of 63- and 65kDa forms, which differ from
one another in the number of sites that are glycosylated. The 65-kDa variant of tPA is glycosylated at Asn-117 of the kringle 1 domain, Asn-184 of the kringle 2 domain, and Asn-448 of the protease domain, while the 63-kDa form is glycosylated at Asn residues 117 and 448 only (2). Cleavage of the peptide bond between Arg-275 and Ile-276 of one-chain tPA by plasmin or some other trypsin-like serine proteases generates two-chain tPA, consisting of two-polypeptide chains, linked by a single disulfide bond (3). The mature secreted form of one-chain tPA, unlike one-chain zymogen forms of most other serine proteases, which show essentially no enzymatic activity, possesses considerable activity (4). However, significant differences do exist between one-chain and two-chain forms of tPA in their enzymatic and other physicochemical properties. Elucidation of these differences has been a focus of much attention to understand the physiological significance for the conversion of one-chain to two-chain tPA (5). Purification of tPA in the absence of a protease inhibitor, such as aprotinin, results in the cleavage of one-chain tPA by endogenous proteases, yielding primarily the two-chain form tPA (6). If aprotinin is present during purification, proteolytic cleavage of one-chain tPA is inhibited and the predominant product is one-chain tPA (6, 7). However, most purified preparations of one-chain tPA are contaminated with variable amounts of two-chain tPA. There is no effective procedure currently available for the removal of two-chain tPA contaminant from one-chain tPA preparations. Consequently, much of the work on enzymatic properties of one-chain tPA has been carried out with preparations contaminated with variable amounts of two-chain tPA. Here 1 describe a simple and effective procedure of affinity chromatography on benzamidine-Sepharose that removes two-chain contaminant and concomitantly separates differentially glycosylated one-chain variants from preparations of one-chain tPA. Some of the data described in this paper have been presented at the Tenth International Congress on Fibrinolysis.
EXPERIMENTAL ‘This work was supported by Research Grant HL38178 from the National Institutes of Health. ’ Abbreviations used: tPA, tissue plasminogen activator; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin. $3.00 0 1991 by Academic Press, of reproduction in any form
02114
29, 1990
Preparations of one-chain tissue plasminogen activator @PA), usually a mixture of 65- and 63-kDa differentially glycosylated forms, contain variable amounts of twochain tPA. There is no effective procedure currently available for removal of the two-chain contaminant from one-chain tPA preparations. In this report, affinity chromatography on benzamidine-Sepharose was investigated for the separation of two-chain from one-chain tPA. Activase, a preparation of recombinant tPA containing 80% one-chain tPA, a mixture of 65- and 63-kDa variants, and 20% two-chain tPA, was applied to a column of benzamidine-Sepharose, equilibrated with 1 M ammonium bicarbonate. Under this condition, both one-chain and two-chain forms of tPA were adsorbed by the column. Addition of 0.1 M arginine to the equilibration buffer led to elution of two-peaks, corresponding to the 65- and 63kDa variants of one-chain tPA. Two-chain tPA remained bound to the column, but could be eluted with sodium acetate buffer, pH 4.0, containing 0.1 M arginine. The present procedure allows rapid and effective removal of two-chain tPA with concomitant separation of 65- and 63-kDa one-chain glycoforms from preparations of onechain tPA. Kinetic analysis for the hydrolysis of D-IkPro-Arg-p-nitroanilide (S-2288) by the highly purified molecular forms of tPA suggests that 63-kDa one-chain tPA possesses 30% higher catalytic efficiency than the 65-kDa variant, while two-chain tPA is 9- or la-fold more efficient than 63- or 65-kDa one-chain tPA, respectively. 0 1991 Academic Press, Inc.
0003-9861/91 Copyright All rights
School, Boston, Massachusetts
PROCEDURES
Material. Activase (Alteplase), a preparation of recombinant tPA, was produced by Genentech (San Francisco, CA). The preparation contained 80% one-chain tPA, a mixture of 65- and 63-kDa forms, and 20% two-chain tPA. A preparation of single-chain tPA, containing 90-95% 373
Inc. reserved.
374
S. SHAUKAT
Fraction
HUSAIN
Number
FIG. 1. Chromatography of Activase (a preparation of one-chain recombinant tPA) on a benzamidine-Sepharose column. The tPA preparation in 1 M ammonium bicarbonate was chromatographed on the column as described in the Results section. Fractions (1 ml) were collected at 7 ml/ h. After the column was washed with the equilibration buffer, elution was performed with 0.1 M arginine in 1 M ammonium bicarbonate at the arrow marked “a” and with 0.1 M sodium acetate, 1 M NaCl, 0.3 M arginine, pH 4, at the arrow marked “b”. single-chain form, derived from mammalian cell culture was obtained from Calbiochem. Chromogenic substrate for tPA, D-Ile-L-Pro-L-Argp-nitroanilide (S-2288), was obtained from Kabi Vitrum, Stockholm, Sweden. Prepacked gel filtration columns, Econo-Pat lODG, were obtained from Bio-Rad, (Richmond, CA). Centricon 10 microconcentrator was obtained from Amicon (Danvers, MA). Other chemicals were obtained from the following sources: 6-aminohexanoic acid coupled to Sepharose 4B (CH Sepharose 4B) and benzamidine-agarose @-aminobenzamidine coupled to agarose via the spacer glycyl-glycine), Sigma; N-ethyl-N-(3dimethylaminopropyl)carbodiimide hydrochloride and 4aminobenzamidine hydrochloride, Aldrich, protein grade Tween 80, Calbiochem. Methods. Benzamidine-Sepharose was prepared by coupling 4-aminobenzamidine to CL-Sepharose in the presence of iV-ethyl-iV’-(3methylaminopropyl)carbodiimide as described by Holmberg et al. (8). The derivative was extensively washed to remove by-products of the reaction as described in the procedure. Polyacrylamide SDS gel electrophoresis of tPA samples, reduced with 20 mM dithiothreitol for 3 min at lOO’C, was performed on 10% slab gels by the method of Laemmli (9). The gels were stained with Coomassie brilliant blue. Amidolytic activity of tPA was determined with 0.5 mM S-2288 in 0.05 M Tris-HCl, 0.15 M NaCl, 20 KIU/ml Trasylol, 1 mg/ml BSA, 0.01% Tween 80, pH 7.4, at 37’C. The increase in absorbance at 406 nm was continuously monitored using a Hewlett-Packard diode array spectrophotometer. For kinetic analysis of the amidolytic activities of various molecular forms of tPA, assays were performed with the substrate concentration varying from 0.0625 to 2 mM. Kinetic parameters were determined from Eadie-Hofstee (10) and Lineweaver-Burk (11) plots using linear regression analyses. RESULTS
Enzyme Purification Activase (50 mg vial, containing 1.7 g arginine, 0.5 g phosphate, and 4 mg polysorbate 80) was reconstituted with 25 ml water. To remove arginine and other salts, an aliquot of the Activase solution (2.5 ml containing 5 mg protein) was passed through Econo-Pat 1ODG column (10 ml bed volume) equilibrated with 1 M ammonium bicarbonate (adjusted to pH 8.0). The protein emerged as a sharp peak in the breakthrough fractions.
The pooled, gel-filtered tPA solution (7.6 ml containing 8.5 mg protein) from two Econo-Pat 1ODG columns was applied at 7 ml/h to a column of benzamidine-Sepharose (1 X 20 cm, 10 ml volume) equilibrated with 1 M ammonium bicarbonate (adjusted to pH 8.0). After washing with one column volume of the equilibrium buffer, elution was started with 1 M ammonium bicarbonate containing 0.1 M arginine (adjusted to pH 8.0). This resulted in the elution of two peaks with activities toward S2288 and plasminogen. Further washing of the column with 0.1 M sodium acetate, 1 M NaCl, 0.3 M arginine, pH 4.0, led to the elution of a third enzymatically active peak (Fig. 1). Fractions 34-46 (Peak l), 57-75 (Peak 2), 47-56 (region between Peaks 1 and 2), and 82-88 (Peak 3) were concentrated in a Centricon 10 concentrator to give solutions with protein concentrations of approximately 1 mg/ml. SDS-polyacrylamide gel electrophoresis of the starting tPA showed that the preparation was a mixture of 80% one-chain and 20% two-chain tPA. The one-chain tPA band comprised two closely spaced 65- and 63-kDa bands (not clearly distinguishable because of overloading in the gel shown). The first peak eluting with 0.1 M arginine-ammonium bicarbonate from the benzamidine-Sepharose column contained 65-kDA onechain tPA, while the closely following second peak contained 63-kDa one-chain tPA. The area between the two peaks was mostly 63-kDa tPA with some 65-kDa variant. The tPA peak eluting with the acetate buffer contained essentially pure twochain tPA (Fig. 2). The purification procedure was repeated with a different preparation of single-chain tPA (Calbiochem) as well as with a commercially available preparation of immobilized benzamidine that has the ligand attached to agarose via glycyl-glycine spacer (Sigma). Chromatography of the single-chain tPA preparation on benzamidine immobilized on Sepharose via aminohexanoic acid spacer resulted in the separation of single-chain and twochain forms of tPA as described for Activase. Chromatography of the tPA preparation on benzamidine attached to agarose via glycyl-glycine resulted in the adsorption of various forms of tPA by the affinity matrix. However, the bound tPA slowly leeched
SEPARATION
OF
ONE-
AND
TWO-CHAIN
FIG. 2. SDS gel electrophoresis of reduced tPA fractions: Protein bands were stained with Coomassie blue. The molecular marker contained phosphorylase (92 kDa), BSA (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa). Lane 1, molecular-weight markers; Lane 2, starting t PA; Lane 3, fractions 34-46 (Peak l), 65-kDa one-chain t PA, Lane 4, fractions 47-56 (region between Peak 1 and 2); Lane 5, fractions 5775 (Peak 2), 63-kDa one-chain tPA; Lane 6, fractions 82-88 (Peak 3), two-chain t PA; Lane 7, molecular-weight markers.
out with the equilibration buffer and application of the argininecontaining buffer caused elution of various forms of tPA in one peak. This suggests that the benzamidine immobilized via the glycyl-glycine spacer binds tPA less tightly than the ligand immobilized through the aminohexanoic acid arm.
Kinetics of Hydrolysis of S-2288 by the Purified TwoChain and 65- and 63-kDa One-Chain Forms of tPA Kinetic constants for the hydrolysis of S-2288 by two-chain tPA and the 65- and 63-kDa variants of one-chain tPA are given in Table I. The values determined from Lineweaver-Burk or Eadie-Hofstee plots are in close agreement. Hydrolysis of the chromogenic substrate by two-chain tPA is characterized by lower K,,, and higher K,,, values than those of the one-chain variants. The resulting catalytic efficiency of two-chain tPA for S-2288 is 12 and 9 times higher than those of the 65- and 63kDa one-chain forms of tPA, respectively. Both K,,, and k,, values of 65-kDa one-chain tPA are higher than the corresponding values of the 63-kDa one-chain variant. The difference in the K,,, is comparatively more than the difference in the k,, values. The catalytic efficiency of 63-kDa one-chain tPA is therefore about 30% higher than that of the 65 kDa variant.
TISSUE
PLASMINOGEN
one-chain tPA. The procedure also results in the separation of 65- and 63-kDa variants of one-chain tPA. The differentially glycosylated variants of tPA have been previously separated by gradient elution from arginineSepharose (12) or lysine-Sepharose (13-15). However, these procedures fail to separate two-chain tPA from onechain tPA. The present method provides a rapid one-step procedure for the removal of two-chain tPA with concomitant separation of one-chain tPA variants. The method provides a comparatively inexpensive source of highly purified 65- and 63-kDa variants of one-chain tPA and the by-product two-chain tPA from clinical preparations. tPA undergoes extensive aggregation and precipitation at concentrations higher than 50 pg per milliliter, except in the presence of chaotropic agents, such as arginine, ammonium thiocyanate, or ammonium bicarbonate that inhibit aggregation. The effectiveness of the benzamidineSepharose column in 1 M ammonium bicarbonate greatly facilitated the present separation procedure. The kinetic efficiencies of 65- and 63-kDa one-chain tPA for S-2288 were found to be 8 and lo%, respectively, of that of two-chain tPA. Earlier figures obtained with mixtures of one-chain 65- and 63-kDa variants contaminated with varying quantities of two-chain tPA suggested catalytic efficiencies of 36% (16), 26% (17), and 17% (18) for one-chain tPA compared to that of two-chain tPA. The present values indicating a larger difference between the activities of one-chain and two-chain tPA reflect a higher degree of enzyme purity. The present investigation also suggests that glycosylation at the Asn-117 residue for the hydrolysis S-2288 affects the kinetic parameters by one-chain tPA. The catalytic efficiency of the 63-kDa one-chain variant, lacking the carbohydrate moiety at Asn-117, is slightly higher than that of the 65-kDa variant. Kinetic parameters for the amidolytic activities of the 63and 65-kDa variants of one-chain tPA have not been previously determined. However, a recent investigation (19) suggests that two-chain tPA unglycosylated at Asn-117 TABLE
tPA is enzymatically active, chroSince one-chain matographic procedures or inhibitor treatment that are usually used to remove active two-chain from inactive one-chain forms of serine proteases are believed to be ineffective for the removal of two-chain tPA from preparations of one-chain tPA. However, this report shows that chromatography of tPA on benzamidine-Sepharose results in an effective separation of two-chain tPA from
I
Kinetic Constants for the Hydrolysis of D-Ile-Pro-Arg-pNitroanilide (S-2288) by Various Molecular Forms of tPA Form
DISCUSSION
375
ACTIVATOR
of tPA
K,,, (mM)
kc., W')
kcat/Km 6.24 + 0.18 (6.4 _+ 0.24)
65-kDa
one-chain
tPA
2.0 (1.8
f 0.1 f 0.1)
12.3 + 0.24 (11.5 +- 0.58)
63-kDa
one-chain
tPA
1.1 + 0.1 (0.93 + 0.1)
8.7 f 0.55 (7.7 * 0.51)
7.7 (8.3
rl: 0.22 + 0.39)
0.40 k 0.02 (0.37 f 0.02)
30.2 zk 0.15 (29.0 z!z 0.15)
76.1 (78.2
+- 3.13 +- 4.1)
Two-chain
tPA
Note. Data are the means of three experiments + SD. The kinetic parameters were calculated from Eadie-Hofstee (10) plot and Lineweaver-Burk (11) plot (values in parentheses).
376
S. SHAUKAT
has higher plasminogen activator activity than the variant with a carbohydrate chain at this residue.
HUSAIN
8.
Holmberg, L., Bladh, Acta 445,215-222.
9. Laemmli,
ACKNOWLEDGMENT The author in performing
Zaidi
for technical
assistance
Lett.
2. Pohl, G., Kallistrom,
M., Bergsdorf, N., Wallen, H. (1984) Biochemistry 23,3701-3707. Ichinose, A., Kisiel, W., and Fujikawa, K. (1984) 412-418.
P., and Jornvall, FEBS
I&t.
1’75,
4. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257,2920-2925. 5. Husain, S. S., Hasan, A. K. H., and Budzynski, A. Z. (1989) Blood 74, 999-1006. 6. Rijken, D. C., and Collen, D. (1981) J. Biol. Chem. 256,7035-7041. 7. Wallen, P., Ranby, M., Bergsdorf, N., and Kok, P. (1981) in Progress in Fibrinolysis (Davidson, Vol. 5, pp. 16-23, Churchill
J. F., Nilsson, Livingstone,
I. M., Astedt, Edinburgh.
B., Eds.),
H., and Burk, B. H. J. (1952)
12. Ranby,
1. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., and Collen, D. (1983) Nature (London) 301, 214-221
Nature
B. (1976)
(London)
D. (1934)
Biochem.
Biophys.
227.680-685.
J. Amer.
Chem.
Sot. 56,658-
666. 11. Hofstee,
REFERENCES
3.
U. K. (1970)
10. Lineweaver,
thanks Mr. Syed Tanweer gel electrophoreses.
B., and Astedt,
M.,
Bergsdorf,
Science N., Pohl,
116, 329-331. G., and Wallen,
13. Einarsson, M., Brandt, J., and Kaplan, L. Actu (1985) 830, l-10. 14 Parekh, R. B., Dwek, R. A., Thomas, J. demacher, T. W., Wittwer, A. J., Howard, N. R., Jennings, M. G., Harakas, N. K., ch.emis0-y 28,7644-7662. 15 Wittwer, A. J., and Howard, S. C. (1990) 4180.
16. Urano, Arch.
P. (1982)
FEBS
146,289-292. (1985)
Biochim.
R., Opendakker, G., RaS. C., Nelson, R., Siegel, and Feder, J. (1989) BioBiochemistry
T., deserrano, V. S., Urano, S., and Castellino, Biochem. Biophys. 270, 356-362.
17. Loscalzo,
J. (1988)
18. Ranby, M., Bergsdorf, 175-183.
J. Clin. Inuest.
Biophys.
82,
1391-1397.
N., and Nilsson,
T. (1982)
29,
F. J., (1989)
Thromb.
19. Wittwer, A. J., Howard, S. C., Carr, L. S., Harakas, Feder, J. (1989) Biochemistry 28, 7662-7669.
4175-
Res. 27, N. K., and