Modified GMP-affinity chromatography for the purification of mutant hypoxanthine phosphoribosyltransferase.
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to IMP and GMP, respectively, in the presence of 5-phosphoribosyl-1-pyrophosphate. Deficiencies of HPRT are associated with neurological abnormalities and gout. A human HPRT variant enzyme failed to bind to a GMP-affinity column under standard purification conditions. We developed a series of predictive tests for designing the affinity chromatography protocol which enabled purification of both normal and variant HPRT. The primary variable for the present variant was a difference in toleration of salt; other aspects recommended for evaluation are assessment of ligand-enzyme affinity, pH optimum, and tolerance of nonspecific ligands for washes. In addition, a method for determining the amount of GMP linked to the column material was developed and consisted of acid hydrolysis and HPLC quantitation of guanine.
Carter-Edwards
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T
Department of Pediatrics, University of Calgary, Alberta, Canada.
Fung
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E
Snyder
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FF
D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D014644:Genetic Variation; D006150:Guanine Nucleotides / Q000138:chemical synthesis; D006157:Guanosine Monophosphate / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000032:analysis / Q000302:isolation & purification*; D008214:Lymphocytes / Q000201:enzymology; D009154:Mutation; D012685:Sepharose / Q000138:chemical synthesis*
D006150:Guanine Nucleotides; D006157:Guanosine Monophosphate; D012685:Sepharose; D007041:Hypoxanthine Phosphoribosyltransferase