An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides

  • A.A Chistov Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya str., GSP-7, Moscow, 117997 Russia
  • A.V. Talanova Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • M.V. Melnikova Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • S.S. Kuznetsova Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • E.F. Kolesanova Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
Keywords: thrombin; adsorption; Bio-Beads SM-2; solid-phase peptide synthesis; p-nitroanilides; chromogenic substrates


Low molecular weight chromogenic thrombin peptide substrates, p-nitroanilides of short peptides protected at their N-terminal amino group, were prepared by solid-phase peptide synthesis on polystyrene-divinylbenzene polymer with trityl groups with preliminary attached p-phenylene diamine moiety. After the cleavage from the resin peptide p-aminoanilides were mildly oxidized to p-nitroanilides with the mixture of potassium sulfate and persulfate. Adsorption onto polymer support Bio-Beads SM-2 with further elution by acetonitrile allowed easy separating peptide p-nitroanilides from the oxidizer and obtaining the thrombin chromogenic substrate preparations with the target substance contents of not less than 95% and yields of 30-40%. Thrombin effectively catalyzed hydrolysis of the prepared substrates with KM and Vmax values of 29-134 mM and 0.03-1/16 mM/s, respectively.


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