US EPA

8218054 

Journal Article 

Effect diabetic drugs on a novel kinetic properties of Glutathione-S-transferase Enzyme( GSTs) from human serum 

Al-Zamely, OMY; Ridha, GAOA 

2016 

0 

Research Journal of Pharmaceutical, Biological and Chemical Sciences
ISSN: 0975-8585 

7 

5 

2070-2087 

English 

Glutathione -S-transferase (GSTs)(EC 2.5.1.18) are dimeric enzyme present in many tissues including lungs, liver and found in mammals, insects, plants, microbes and divided into four classes including alpha,mu,pi,theta. GSTs are important phase II drug-metabolizing enzymes that a major role in protecting cells from toxic . This study attempt isolate GST enzyme from serum human .The partial purification of glutathione -S-Transferase were done using DEAE-Cellulose, Then purification steps include precipitation by ammonium sulfate 70%, then using dialyzed by dialyze tube cut off number (10-14KD)overnight against the phosphate buffer pH=7 and further purified with DEAE-Cellulose column chromatography with a(55.14) purification folds then the glutathione -s-transferase enzyme purified also from serum human by using pre-packed column affinity chromatography by containing glutathione Sepharose 4% and compare activity between two separation ways the activity of GSTenzyme result from affinity column were more by using ion exchange column and found that enzymatic efficiency of GST by using affinity column (100%) while at using ion-exchange chromatography (55.14%) . The molecular weight of enzyme was determined by using SDS-PAGE analysis by using enzyme GST result from ion-exchange chromatography and affinity chromatography found (28KD). This study involved also some kinetic studies of GST, maximum activity for GST enzyme was obtained using 22.463mmol/l of 1-chloro-2,4-dinitrobenzene (CDNB)as substrate, the enzyme showed maximum activity at 35°C and optimum pH at 6.8 and time at 12 minutes in incubation at 35°C . Using Lineweaver - Burk plot the maximum velocity (Vmax ) and Michaelis constant Km were (11.12mmol/liter) and Vmax (1.254μmol/min) respectively .The thermodynamic constants of activation, (ΔH, Ea, Δs, and ΔG) were determined by using Arrhenius plot and found to be(35.866KJ/ mol,31.73KJ/mol,12.06 KJ/mol.K, -3.559 J /mole.K) respectively .Metformin and Dionial inhibition on the GST activity were found that, this lead to drugs effect on enzyme GST.Where showed result that non comparative inhibitor at metformin and showed that comparative inhibitor at dionial. 

Glutathione-S-transferase; Human serum; Kinetic properties