Open AccessAn Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides
Author(s) -
Alexey A. Chistov,
A.V. Talanova,
М. В. Мельникова,
S.S. Kuznetsova,
Е. Ф. Колесанова
Publication year - 2018
Publication title -
biomedical chemistry: research and methods
Language(s) - English
Resource type - Journals
ISSN - 2618-7531
DOI - 10.18097/bmcrm00057
Subject(s) - chemistry , peptide , divinylbenzene , potassium persulfate , hydrolysis , moiety , substrate (aquarium) , polymer chemistry , polystyrene , tosyl , polymer , combinatorial chemistry , chromatography , nuclear chemistry , polymerization , organic chemistry , copolymer , biochemistry , styrene , oceanography , geology
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.