Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase

P. Ascenzi, G. Amiconi, E. Menegatti, M. Guarneri

Research output: Contribution to journalArticle

Abstract

A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu1, Lys77-, Val442-, and Val561-plasmin, as well as of active human Glu1-, Lys77-, Val442- and Val561-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu1-, Lys77-, Val442- and Val561-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu1-, Lys77-, Val442- and Val561-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu1-, Lys77- Val442- and Val561-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10-3 μM.

Original languageEnglish
Pages (from-to)183-190
Number of pages8
JournalClinical Chemistry and Enzymology Communications
Volume3
Issue number2-3
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Clinical Biochemistry

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