PURIFICATION AND PROPERTIES OF GLUTATHIONE S-TRANSFERASE

Abstract

Glutathione S-transferase (E.C.2.5.1.18) catalyzes the nucleophilic attack of the sulfur atom of glutathione on electrophilic groups in a second substrate. The enzymes occur ' abundantly in most forms of life investigated and are generally considered to serve in the intracellular detoxification of mutagens, carcinogens, and other• noxious chemical substrates.

In the present work, GSH S-transferase from S. haematobium adult worms was prepared. By GSH-affinity column using a pH 9.6 buffer containing GSH, two forms designated as bound and unbound fractions were separated. Using HPLC chromatofocusing with a shallow gradient from pH 8.0 to 6.0, the bound fraction was further separated into two forms designated as ShGST-1 and ShGST-2. Different techniques were applied separately to purify the GSH-affinity unbound fraction. We used different columns in different batches of purification. First, we used phenyl agarose column with a stepwise gradient of ammonium sulfate from 1.5 to 0.1 M. No activity was detected in all collected fractions using

CDNB as substrate. We then used a CM-Sepharose columr::t and a pH 6.0 potassium phosphate buffer, all the activity appeared in the void volume of the column which means that the enzyme did not bind to the cation exchanger. The same results were obtained even when the pH of the buffer was changed.