Purification and characterization of glutathione S-transferase enzyme from chicken gizzard

In this study, which was carried out to purify the glutathione S-transferase (GST; EC 2.5.1.18) enzyme from chicken gizzard, glutathione-agarose affinity chromatography and ammonium sulfate precipitation were used as methods. As a result of the study, 69.17 EU/mg protein was purified 1441 times, achieving 85.42% yield with specific activity. SDS-PAGE study was performed to obtain single-band image, determine subunit molecular masses and check enzyme purity. At the end of the study, the image was obtained as a single band. Characterization studies were carried out for the Chicken gizzard GST enzyme, whose subunit molecular mass was determined to be approximately 25.66 kDa. With the characterization studies, the optimum ionic strength was determined as 300 mM in Tris-HCl buffer. The optimum pH was found to be pH= 7.5 in K-fosfat buffer and the optimum temperature was 40 ºC. Stable pH was obtained as pH= 7.0-7.5 in K-fosfat buffer. KM and Vmax values were determined for the purified enzyme. The Lineweaver-Burk chart was used to determine the values. While KM was determined as 11.4761 mM in CDNB substrate, Vmax was determined as 0.5837 EU/mL. For GSH substrate, KM was found to be 11.1348 mM and Vmax was 0.75205 EU/mL.