Abstract

Si fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-lebelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their α and β chains by preparative reversed-phase HPLC using a macroporous C 4 support. Phosphate analyses indicate that the β chains of II, III and IV each contain one phosphate per chain while the β chains of V and VI wach contain two phosphates. The α chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the β chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the α chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertable and that they correspond to the PLP 2-Hb species and component V is Benesch's PLP 4-Hb [J. Biol Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per α chain and similarly VI is V with monolabelled α chains. Both IV and VI are hitherto unknown pyridoxal 5'-phosphate derivatives of hemoglobin. The oxygen affinities of the components were determined and were found to decrease with increases in the extent of pyridoxal '-phosphate labelling. This effect, however, reaches a maximum in V since VI which contains 6 mol of phosphate per mol of hemoglobin has a higher affinity than V.

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