The action of thrombin on peptide p-Nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction condtions

Richard Lottenberg, Julie A. Hall, Morey Blinder, Ellen P. Binder, Craig M. Jackson

Research output: Contribution to journalArticle

108 Scopus citations

Abstract

Kinetic parameters for the action of bovine α-thrombin on 24 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kcat Km, ranges from 3.3 · 101 to 1.1 · 108 M-1 · s-1 for the poorest and the best substrates, respectively. The best substrates for thrombin were identified as those with arginine in the P1 position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effects of NaCl, CaCl2 and poly(ethylene glycol) concentrations, pH and temperature on the action of thrombin on selected substrates have been assessed. A source of large systematic error in thrombin concentration estimates was identified as resulting from adsorption losses. These losses were eliminated by inclusion of poly(ethylene glycol) in dilution and reaction buffers.

Original languageEnglish
Pages (from-to)539-557
Number of pages19
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume742
Issue number3
DOIs
StatePublished - Feb 15 1983

Keywords

  • Hydrolysis
  • Peptide p-nitroanilide
  • Poly(ethylene glycol)
  • Substrate selectivity
  • Thrombin

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