Extraction, Purification and Kinetic Study of Lactate Dehydrogenase of Male Chicken from Ebocha-oil Exploration Area, Nigeria

Abstract

Aim: This study focused on the extraction, purification and kinetic studies of lactate dehydrogenase of male chickens from Ebocha oil exploration area, Imo state, Nigeria. Methods: Twenty-one apparently healthy mature (6-9 months) male chickens (Gallus domesticus) from Ebocha oil exploration area, Imo State, Nigeria were screened for lactate dehydrogenase activity, thus accessing the level of chronic cell exposure to gas flaring. Their thigh muscle tissues were severed and investigated for lactate dehydrogenase activity using the standard method and sodium pyruvate as the substrate. Lactate dehydrogenase was isolated and purified by ammonium sulphate precipitation, desalted by dialysis and then gel filtration. Results: The enzyme activity increased with advancement in the purification steps and was maximum using dialysis. The values for the lactate dehydrogenase activities were 103.43±3.27 U/L, 279.50±5.38 U/L, 318.16±13.08 U/L, 100.47±2.59 U/L, with a purification fold of 1, 3.7, 6.24 and 2.55 for the purification steps respectively. Also, the values of the protein concentrations were 0.071 mg/ml, 0.050 mg/ml, 0.035 mg/ml and 0.027 mg/ml (values for the crude enzyme, ammonium sulphate precipitation, dialysis and gel filtration respectively). The enzyme showed optimal activity at pH range of 5.5-6.5 and temperature of 30ºC-40ºC. Using sodium pyruvate as the substrate, with a fixed enzyme volume, an increase in the concentration of substrate resulted in increase in enzyme activity until a saturation point 0.3mM was reached. The apparent Km and Vmax values obtained were 0.01 mM and 0.12 U/mg/min. The Lineweaver-burk plot of the partially purified enzyme gave real Km and Vmax values of 0.20 mM and 0.16 U/mg/min respectively. Conclusion: Partial purification procedures and biochemical properties of lactate dehydrogenase, from the muscle tissues of male chickens of Ebocha origin, gives room for more investigation on the metabolic shift caused by chronic exposure of the environment, humans and livestock to gas flaring and petroleum exploration.