Selective elution of subunit 4-containing rat liver glutathione S-transferases using affinity chromatography

Glutathione S-transferase (GST) isoenzymes of rat liver cytosol were purified from a S-hexylglutathione Sepharose affinity column without using S-hexyl-glutathione for elution. The method involves an initial elution of GSTs 3-4 and 4-4 from the affinity matrix with 10 mM glutathione in 50 mM Tris-HCl (pH 7.0), followed by the elution of GSTs 1-1, 1-2, 2-2 and 3-3 with 10 mM glutathione in 50 mM Tris-HCl (pH 9.6). These two fractions were then chromatofocused simultaneously to separate the individual GST dimers. The ampholytes used in this process were subsequently removed by ultrafiltration, as the standard techniques (Sephadex G25 column, and dialysis) were not effective. This method provides improved yields and reduced losses of GST activity, by halving the preparation time.