Modified GMP-affinity chromatography for the purification of mutant hypoxanthine phosphoribosyltransferase

• Published in: Analytical biochemistry Vol. 178; no. 1; pp. 148 - 152
• Main Authors: Carter-Edwards, Therese, Fung, Ernest, Snyder, Floyd F.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.04.1989; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cells, Cultured; Chromatography, Affinity - methods; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genetic Variation; Guanine Nucleotides - chemical synthesis; Guanosine Monophosphate - chemical synthesis; Hydrogen-Ion Concentration; Hypoxanthine Phosphoribosyltransferase - analysis; Hypoxanthine Phosphoribosyltransferase - isolation & purification; Lymphocytes - enzymology; man; Mutation; Sepharose - chemical synthesis; Transferases; Chromosomal aberration; Human; Purification; Abnormal X chromosome; Enzyme; Environmental factor; Hypoxanthine phosphoribosyltransferase; Lesch Nyhan syndrome; Purine base; GMP; Guanine; Affinity chromatography; Clinical biology; Abnormal chromosome; Hypoxanthine; Nucleoside; Mutation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(89)90371-0

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.