A single-step separation of the one- and two-chain forms of tissue plasminogen activator.
Preparations of one-chain tissue plasminogen activator (tPA), usually a mixture of 65- and 63-kDa differentially glycosylated forms, contain variable amounts of two-chain 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 63-kDa 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 one-chain tPA. Kinetic analysis for the hydrolysis of D-Ile-Pro-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 12-fold more efficient than 63- or 65-kDa one-chain tPA, respectively.
Husain
S S
SS
Department of Anesthesia, Massachusetts General Hospital, Boston 02114.
D001120:Arginine; D001550:Benzamidines; D002846:Chromatography, Affinity; D002863:Chromogenic Compounds / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*
D001550:Benzamidines; D002863:Chromogenic Compounds; D009842:Oligopeptides; D011994:Recombinant Proteins; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D001120:Arginine; D010959:Tissue Plasminogen Activator; C032157:benzamidine