دراسات حركية ثرموديناميكية لانزيم الكلوتاثيون الناقل المنقى جزئيا من مصل دم الانسان مع تاثير بعض ادوية علاج السكري
Author name:
غفران عبد عمران عبد الرضا محمد الخفاجي
Supervisor name:
عودة مزعل ياسر الزاملي
General topic:
Chemistry
Specific topic:
Chemistry
Degree:
Master
University:
University of Babylon - College Of Science
Language:
English
University location:
Babylon
First pages:
25T1547 - p.pdf
Abstract:
This study involved (30) Person healthy (15males & 15 femals) the ages were between (20 - 25) years were obtained on samples of students from the university of Babylon / College of Science in Babel province, Iraq has been selected as control group without chronic disease and without smoking.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%, The specific activity was 0.244 U/mg protein and purification degree 1.07 folds and stepwise of Di ethyl amino ethyl - cellulose chromatography and further purified with DEAE - Cellulose column chromatography. The enzyme was apply on DEAE - Cellulose (1.5×30cm) and flow rate 1ml/min and The specific activity was 0.314 U/mg protein with recovery 55.14% and purification degree 1.50 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 specific activity between two separation ways the activity of GST enzyme result from affinity column were more by using ion exchange column and measured of enzyme activity at 340nm by using CDNB as substrate of GST enzyme .The electrophoresis of the partial from human serum from ion - exchange chromatography and affinity chromatography in polyacrylamide gel was found that it was one protein band by using SDS - PAGE analysis by usingenzyme GST result from ion - exchange chromatography 27.7KD 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 activityat 35 and optimum pH at 6.8 and time at 12 minutes in incubation at 35 . 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 , were determined by using Arrhenius plot and found to be (35.906kJ. mol - 1, 31.73kJ.mol - 1, - 1.729 kJ.mol - 1.k ,551.40 kJ.mol - 1.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 .
