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

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Published: Nov 7, 2018
DOI: https://doi.org/10.18097/BMCRM00057
Keywords:
thrombin; adsorption; Bio-Beads SM-2; solid-phase peptide synthesis; p-nitroanilides; chromogenic substrates

Main Article Content

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

Abstract

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.

Article Details

How to Cite
Chistov, A., Talanova, A., Melnikova, M., Kuznetsova, S., & Kolesanova, E. (2018). An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides. Biomedical Chemistry: Research and Methods, 1(4), e00057. https://doi.org/10.18097/BMCRM00057
Issue
Vol. 1 No. 4 (2018)
Section
EXPERIMENTAL RESEARCH

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