Журнал аналитических и биоаналитических методов

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Purification, Determination Molecular Weight and Study Kinetic Properties of G6PD from Diabetes Patient

Abdulkader Rasheed W, Firas Maher T and Akeel Al-Aisse H

This study was conducted to purification G6PD enzyme from diabetic patients by using simple and cheap method the technique gel filtration on Sephadex G100 and determine molecular weight of enzyme and compare it with true molecular weight of enzyme and determine kinetic constant (Km, Vmax) and study the effect of temperature and substrate and pH and known the best condition to give optimum work of enzyme. study contain (60) patients with diabetes and (60) control Glucose and activity of G6PD were measured and the enzyme precipitated by Ammonium Sulfate with concentration (75%) and purification enzyme gel filtration on Sephadex G-100 with dimensions (1.5 × 30) cm and using the buffer solution from (Tris-HCl) at pH 8.2 to isolate the enzyme and determine molecular weight with same method. Specific activity was calculated (21.5 UI/mg), total activity (706.8 UI), number of purification (3.45) enzyme yield (23.188%) and enzyme activity (17.67 UI/ml). and the molecular weight was calculated with using same technique (57.82) kD. Effect of increased concentration of substrate on enzyme activity and found the activity increase with increase substrate and amount constant level not change however increase of concentration of substrate when drawing relation between activity and concentration of substrate format appear exchange excess and after study effect of pH found the optimum value (8.4) and study effect of temperature on activity found (38 C) the optimum temperature. Study of kinetic constant was done and the and the Michaelis-Menten (Km) value was (3.8 mM) and Vmax value (8 IU/ml).