Purification of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from [formula omitted] using affinity chromatography: Resolution of monomeric and dimeric forms

Abstract

Anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) as the unaggregated enzyme, has been purified to 90–95 percent homogeneity using affinity and ion-exchange chromatography. The affinity matrix consisted of anthranilate coupled to succinylamidohexamethylimino-Sepharose via its free amino group. Electrophoresis of the purified PRT on gels of varying polyacrylamide concentrations resolved a faster-moving minor band at concentrations above 8 percent. Both the major and minor bands were found to possess PRT activity. The minor band was found to have a molecular weight one-half that of the major band. Electrophoresis of the enzyme in the presence of sodium dodecyl sulfate resulted in almost complete conversion to the lower molecular weight form.