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 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.
Schneedorf
J M
JM
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
Santoro
M M
MM
Mares-Guia
M
M
D000596:Amino Acids / Q000737:chemistry*; D011232:Chemical Precipitation; D004396:Coloring Agents / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D013783:Textile Industry; D014361:Trypsin Inhibitors / Q000737:chemistry*
D000596:Amino Acids; D004396:Coloring Agents; D014361:Trypsin Inhibitors