A spectrophotometric assay for the determination of the catalytic efficiency of plasminogen activators using a slowly hydrolyzed plasmin substrate

Maria Jose M. Castro, I. Barry Kingston, Stephen Anderson

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

A simple spectrophotometric assay for the determination of the catalytic efficiency and activity of plasminogen activators is presented. The assay system contains activator, plasminogen, and the chromogenic substrate N-benzoyl-L-arginine-p-nitroanilide (BAPA). Plasmin production is monitored continuously by the hydrolysis of BAPA under non-steady-state, first-order conditions with respect to plasminogen. Apparent catalytic efficiency constants are calculated from the values obtained for the apparent first-order rate constant of activation. The results obtained with the present method were compared with the catalytic efficiency determined through the measurement of kcat and Km, using a different system, under steady-state conditions. Tissue plasminogen activator in the absence and presence of fibrinogen and high-molecular-weight urokinase were used as model activators. Potential applications are discussed.

Original languageEnglish (US)
Pages (from-to)225-231
Number of pages7
JournalAnalytical Biochemistry
Volume226
Issue number2
DOIs
StatePublished - Apr 1995

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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