Inhibition of chymotrypsin by heparin cofactor II.

Abstract

Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. We have recently determined that the reactive-site sequence (P1-P'l) in heparin cofactor II is Leu-Ser (9). Therefore, the reactive-site sequence in heparin cofactor II resembles that found in a1-antichymotrypsin (10) but not that in antithrombin III, which is Arg-Ser (11). Chymotrypsin Aa has a substrate specificity primarily directed toward P1 amino acid residues that have large or aromatic hydrophobic side chains (12). The present investigation was undertaken to determine whether or not heparin cofactor II inhibits chymotrypsin and if the reaction rate is enhanced by glycosaminoglycans. We report here that heparin cofactor II inhibits chymotrypsin more rapidly than thrombin, but the reaction rate is not affected by glycosaminoglycans. EXPERIMENTAL PROCEDURESMaterials. Human plasma heparin cofactor II was isolated as described (13). Chymotrypsin Aa, heparin (ammonium salt), dermatan sulfate, and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were obtained from Sigma. Nutritional Biochemicals supplied N-acetyl-a-azaphenylalanine p-nitrophenyl ester; Polybrene and 2,3-butanedione were purchased from Aldrich. All buffers used in this study contained 0.1% (wt/vol) polyethylene glycol (Mr = 8000) to limit protein adsorption to various surfaces.Enzyme and Inhibitor Assays. Chymotrypsin activity was measured by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (14). Heparin cofactor II activity was determined by measuring the rate of chymotrypsin inhibition in the absence and presence of heparin or dermatan sulfate. Stock chymotrypsin solutions (100 AM) were dissolved in 1 mM HCl and kept at 4°C. Inhibition reactions were started by adding chymotrypsin (final concentration, -15-35 nM) to a solution containing heparin cofactor II (final concentration, about 0.05-2 ,uM) in 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl. Portions (0.1 ml) were removed at intervals, added to a substrate solution (0.8 ml) containing 100 AM succinyl-AlaAla-Pro-Phe-p-nitroanilide in 50 mM triethanolamine acetate, pH 8.0/100 mM NaCl, and incubated at ambient temperature. Polybrene (0.5 mg/ml) was included in the substrate solution when inhibition assays were performed in the presence of heparin or dermatan sulfate. Substrate hydrolysis was terminated after a suitable time, usually 3-5 min, by the addition of glacial acetic acid (0.1 ml). pNitroaniline released during the assay was measured at 400 nm and the amount of substrate hydrolyzed was proportional to the chymotrypsin concentration. Antithrombin activity of heparin cofactor II was determined as described (13,15).Under the reaction conditions used in which heparin cofactor II concentration was in excess of the chymotrypsin concentration, apparent pseudo-first-order kinetics was followed. Under these circumstances, the rate of disappearance of free chymotrypsin (E) may be described by ln(E/Eo) = -k2(IO) t, [11 where the residual activity (%) is E/Eo at time t, I0 is the initial concentration of heparin cofac...

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Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

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Heparin Cofactor II Modulates the Response to Vascular Injury

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