Biochemical and Kinetic Study for the Partial Purified Thioredoxin Reductase from Healthy Human Serum

Abstract

The research included the separation of thioredoxin reductase (TrxR) from human blood serum using different biochemical techniques. Two proteinous peaks had been isolated by gel filtration using sephadex)G-50) and three proteinous peaks isolated from sephadex (G-100) that produced by ammonium sulphate precipitation (65%). The approximately molecular weight of the isolated protein as a source of enzyme using gel filtration chromatography (G-100) was (118059 + 925) Dalton.

The results showed two peaks of enzyme when using ion exchange-type DEAE-cellulose and when using electrophoresis technique  type SDS-PAGE indicated the presence of two bands from peak of separation column type sephadex G-100 and one band of each peak, resulting from the ion exchange process with molecular weights of 58231 +490 Dalton.

The results showed that the optimum conditions of purified enzyme from human serum was at (200 µg/ml) of protein as a source of the enzyme using (0.4 mol/l) citric acid-Na₂HPO₄ buffer solution at pH (7) act for (25) minutes at (40°C). Using Line Weaver-Burk plot, the values of maximum velocity (V_max) and Michaelis constant (K_m) were found to be (0.182 µmol/ min) and (0.013 mol/l) respectively using 5,5^-- dithio bis (2-nitrobenzoic acid) (DNTB) as a substrate. Finally, this was, also, involved the study of the effect of some chemicals and drugs on the enzyme activity. The results showed that gundinium - HCl is uncompetitive inhibitor, while bromoacetic acid is noncompetitive inhibitor and zinc sulphate is a competitive inhibitor for the enzyme at different concentrations of inhibitors, but dexathamazone was an activator to the enzyme.