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

• Published in: Brazilian journal of medical and biological research Vol. 31; no. 9; pp. 1105 - 1111
• Main Authors: Schneedorf, J M, Santoro, M M, Mares-Guia, M
• Format: Journal Article
• Language: English
• Published: Brazil Associação Brasileira de Divulgação Científica 01-09-1998
• Subjects: Amino Acids - chemistry; BIOLOGY; Chemical Precipitation; Coloring Agents - analysis; flavianic acid; Hydrogen-Ion Concentration; MEDICINE, RESEARCH & EXPERIMENTAL; textile dyes; Textile Industry; tripsin inhibition; Trypsin Inhibitors - chemistry; flavianic acid; tripsin inhibition; textile dyes
• ISSN: 0100-879X; 1414-431X; 1414-431X; 0100-879X
• DOI: 10.1590/S0100-879X1998000900001

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 beta-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using alpha-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of beta-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 microM; kinetic parameters for the substrate hydrolysis were: Km = 32 microM, 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.