A NEW HEMOGLOBIN-VARIANT FOUND DURING HB A(1C) MEASUREMENT - HB HOKUSETSU [BETA-52(D3)ASP-]GLY]

A new beta chain variant was accidentally found through the assay of H b A(1c) in a diabetic patient. The variant was detected by polyacrylam ide gel isoelectrofocusing and electrospray ionization mass spectromet ry. For sequence determination; globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liqu id chromatography connected to electrospray ionization mass spectromet ry, An abnormal beta T-5 peptide was found by reconstructed selected i on monitoring. The collision-induced dissociation spectrum of an ion d erived from the abnormal beta T-5 peptide revealed a new substitution, [beta 52(D3)Asp-->Gly], named Hb Hokusetsu. The sequence was confirme d with an automatic sequencer using peptides isolated by reversed phas e high performance liquid chromatography. Amplification of the beta-gl obin exon 2 and nucleotide sequencing revealed a G (A) under bar T-->G (G) under bar T mutation in codon 52 corresponding to an Asp-->Gly re placement. Electrospray ionization mass spectrometry analysis of the h emolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal st ability in the isopropanol test. Oxygen equilibrium studies on the pat ient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.