A Simple and Rapid Method for Hemoglobin Removal From Mammalian Tissue Cytosol by Zinc Sulfate and Its Application to the Study of Glutathione Transferase

Abstract

Summary: Glutathione transferase (GST), a cytosolic enzyme responsible for conjugation of glutathione (GSH) with electrophiles, was found to be inhibited by hemoglobin (Hb). The IC50 values of 14 and 45 nu M for Hb were estimated using the affinity purified human term placental and rat hepatic GSTs, respectively. A very simple, rapid, and highly reproducible method that uses zinc sulfate was designed to remove Hb from tissue cytosol without affecting GST. Directly added 500 nu M zinc sulfate did not cause statistically significant inhibition (5-7%) of the affinity purified GST from the human liver, human placenta, and rat liver. A treatment with 500 nu M zinc sulfate was found to be optimal to remove 95% of Hb from the cytosol isolated from the rat liver and human term placenta. Two independent methods to quantitate Hb content gave essentially similar results. The experimental data suggest that a failure to remove Hb from cytosol can lead to about 25% underestimation of total GST content in the rat liver. The zinc sulfate treatment of the rat liver cytosol increased specific activity of GST by 2.5-fold toward 1-chloro-2,4-dinitrobenzene (CDNB) and decreased the Km value from 0.67 to 0.46 mM for CDNB. The specific activity of rat liver cytosolic GST toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, 4-nitropyridine-N-oxide, and ethacrynic acid also increased by 30-60%. The sensitivity of rat liver GST isozymes toward all the inhibitors tested increased by 50-100%. Collectively, the evidence suggests that the zinc sulfate treatment, which efficiently eliminates Hb from tissue cytosol, does not alter structural and functional characteristics of the rat liver GST isozymes. [K]Key [K]Words: glutathione transferase, glutathione transferase inhibition by hemoglobin, hemoglobin removal from tissue cytosol, human liver glutathione transferase, placental glutathione transferase, rat liver, zinc sulfate.

Keywords: :

• [K]Key [K]Words: Glutathione Transferase, Glutathione Transferase Inhibition By Hemoglobin, Hemoglobin Removal From Tissue Cytosol, Human Liver Glutathione Transferase, Placental Glutathione Transferase, Rat Liver, Zinc Sulfate.