Affinity chromatography of hepatic glutathione S-transferases on omega-aminoalkyl sepharose derivatives of glutathione.
Rat liver glutathione S-transferases (RX: glutathione R-transferase, EC 2.5.1.18) were found to adsorb S-carbamidomethyl glutathione linked to Sepharose CL-4B via lysyl or aliphatic diamine spacers of various carbon chain lengths (-NH-(CH2)n-NH-, n = 2, 4, 5, 6, 8 and 10). Proteins were eluted specifically by reduced glutathione. The affinity of the enzymes for the adsorbent increased with increase in the carbon chain length of aliphatic diamine spacers used. Adsorbent having a free carboxyl group within the spacer moiety had high capacity and was specific for glutathione S-transferases. The transferases were specifically eluted from the column in high yield by low concentrations of glutathione. Enzymes purified by the lysyl spacer adsorbent were homogeneous in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and contained most of the hepatic glutathione S-transferase isozymes in isoelectric focusing. Oxidized glutathione and S-methyl glutathione were equally effective as reduced glutathione in eluting glutathione S-transferases from the adsorbent, but gamma-glutamylcysteinylglycineamide or gamma-glutamylcysteinylglycine-1-methyl ester were not effective. These data suggested that the free carboxyl group of glycyl moiety of glutathione might also be important for the specific binding of the transferases to this adsorbent.
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives; D005982:Glutathione Transferase / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008297:Male; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism*
D007527:Isoenzymes; D011134:Polysaccharides; D012685:Sepharose; D005982:Glutathione Transferase; D005978:Glutathione