High-performance liquid chromatographic evaluation of pyridoxyl 5'-phosphate hemoglobin derivatives produced by different reduction procedures

Pyridoxylated hemoglobin derivatives have been studied by many investigators. In this study hemoglobin A0 rather than stroma-free hemoglobin was used as a starting material in order to reduce the number of proteins to A0 and A1c. Derivatives were characterized using a Synchropak Q300 strong anion-exchange column, a PolyCAT A weak cation-exchange column and a VYDAC reversed-phase high-performance liquid chromatographic column. Resulting peak profiles of these two ion-exchange separations demonstrated enhanced resolution and showed the presence of pyridoxylated hemoglobin products not previously described. We compared products from the reduction of these Schiff base derivatives using either sodium borohydride or sodium cyanoborohydride reduction procedures. The sodium cyanoborohydride reduction method produced a lower percentage of products with multiple-site pyridoxylation modifications than the sodium borohydride reduction process.