FLAVIANATE, AN AMINO-ACID PRECIPITANT, IS A COMPETITIVE INHIBITOR OF TRYPSIN AT PH-3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecu le of opposite charge in acid solutions, in a process of reversible de naturation that can be utilized :for protein fractionation. In order t o 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 ou t using alpha-CBZ-L-Lys-p-nitroyhenyl ester as substrate which was add ed to 50 mM sodium citrate buffer, pH 3.0, containing varying concentr ations of a-trypsin and dye. The reaction was recorded spectrophotomet rically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equati ons. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 mu M; kinetic parameters for the substrate hydrolysi s were: Km = 32 mu M, and kcat = 0.38/min. The competitive character o f 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.