Determination of active human urinary kallikrein and human urokinase (M(r) 33,000 and M(r) 54,000 species) concentration in the presence of cognate serine proteinase

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

Research output: Contribution to journalArticlepeer-review

Abstract

A spectrophotometric method for the determination of active human urinary kallikrein concentration, in the presence of human urokinase (M(r) 33,000 and M(r) 54,000 species), as well as of active human urokinase (M(r) 33,000 and M(r) 54,000 species) concentration, in the presence of human urinary kallikrein, is reported. This rate 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 urinary kallikrein, in the presence of human urokinase (M(r) 33,000 and M(r) 54,000 species) selectively inhibited by the p-carbethoxyphenyl ester of the ε-guanidino-caproic acid methanesulphonate, and by human urokinase (M(r) 33,000 and M(r) 54,000 species), in the presence of human urinary kallikrein selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor). The active enzyme concentration has been estimated, under conditions where [S]0 > >K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E]0) taking into account the proportionally constant k(cat) (i.e., V = k(cat)·[E]0). The minimum active human urinary kallikrein and human urokinase (M(r) 33,000 and M(r) 54,000 species) concentration which may be evaluated from the analysis of the experimental data corresponds to 3.8 x 10-3 μM and 1.4 x 10-2 μM, respectively.

Original languageEnglish
Pages (from-to)79-85
Number of pages7
JournalClinical Chemistry and Enzymology Communications
Volume2
Issue number2
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Clinical Biochemistry

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