Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Brazilian Journal of Medical and Biological Research

J.m. Schneedorf¹  M.m. Santoro¹  M. Mares-guia¹ 

[1] ,Universidade Federal de Minas Gerais

关键词: flavianic acid;    tripsin inhibition;    textile dyes;   

DOI  :  10.1590/S0100-879X1998000900001

来源: SciELO

【 摘 要 】

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

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