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Eur. J. Mass Spectrom. 1, 195 - 201 (1995) |
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Characterization of new amino-terminal blocking groups in the normal human adult hemoglobin Hb A1b | ||
Danielle Promé and Jean-Claude Promé |
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ABSTRACT: | ||
Hemoglobin A1b is a minor component (0.5%) of human hemoglobin in the normal adult. It was separated from the main constituent by ion exchange chromatography and further split up into subfractions by IRC-50 chromatography. Hb A1b had been shown previously to contain b-pyruvylated hemoglobin, but several minor components were detected at the trailing edge of the chromatographic peaks. These fractions were chromatographed in 6M urea, which allowed chain splitting and separation into several components. Electrospray mass spectrometry showed that all but one of these fractions were still complex mixtures. Each one was cleaved by trypsin, the resulting peptides were separated by reverse phase high-performance liquid chromatography (HPLC) and analyzed by fast atom bombardment mass spectrometry. By comparison with the normal chain, several abnormal peptides were detected. Their sequence was deduced from comparison of their collision-induced decomposition (CID) spectrum with that of normal peptides. Fragment ions containing the carboxyl end of the molecule identified them as T1 peptides from the a or b chains. In contrast, all ions containing the amino terminus were shifted up by the presence of N-blocking groups. These groups were identified as acetyl, pyruvyl, carbamyl, lactyl, glyceryl and glycosyl residues in the b-T1 peptides, and as formyl, acetyl and carbamyl residues in the a-T1 peptides. These post- translational modifications are likely to be due to the reaction of hemoglobin with chemically reactive metabolites inside the red cells. In vitro incubation of pyruvate with hemoglobin leads to the formation of pyruvylated hemoglobin. The CID-MIKE spectrum of the pyruvylated b-T1 peptide is identical to that from Hb A1b. | ||
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