使用不同底物和抑制剂评估人凝血酶的抑制作用。纤维蛋白肽结合相互作用的表征。

Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction.

作者信息

Gorman J J

出版信息

Biochim Biophys Acta. 1975 Dec 15;412(2):273-82. doi: 10.1016/0005-2795(75)90041-0.

PMID:1191680

Abstract

The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.

摘要

已使用纤维蛋白原、N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺、N-α-苯甲酰基精氨酸对硝基苯胺、N-α-苄氧羰基酪氨酸对硝基苯酯和对硝基苯乙酸评估了人凝血酶的活性：与N-α-苯甲酰基精氨酸对硝基苯胺和对硝基苯乙酸相比，发现N-α-苄氧羰基酪氨酸对硝基苯酯和N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺的水解速率增加。苯甲基磺酰氟和N-α-甲苯磺酰-L-赖氨酸氯甲基酮对每种底物的活性具有相同程度的抑制作用。如用N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺和N-α-苄氧羰基酪氨酸对硝基苯酯所示，在苯甲基磺酰氟反应混合物中加入N-α-甲苯磺酰基精氨酸甲酯可保护酶免受抑制。N-乙酰咪唑对纤维蛋白原、N-α-酪氨酸对硝基苯酯的活性有不同程度的抑制作用。用中性羟胺可使对N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺的抑制作用完全逆转，而对纤维蛋白原的凝血活性仅部分恢复。人纤维蛋白肽A抑制对N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺和N-α-苄氧羰基酪氨酸对硝基苯酯的活性作用。纤维蛋白肽A对N-α-苯甲酰基苯丙氨酰缬氨酰精氨酸对硝基苯胺的抑制模式为非竞争性（K1 = 3.02×10⁻⁵ M），而N-α-甲苯磺酰基精氨酸甲酯是该底物的竞争性抑制剂（K1 = 2.6×10⁻⁵ M）。这些研究表明人凝血酶上纤维蛋白原有不止一个结合域。