Fluorescence and biochemical characterization of glycated hemoglobin

Isoelectric focusing (IEF) of glycated hemoglobin (GHb) was carried out in ultra-thin polyacrylamide gels to separate the hemoglobin-advanced glycation endproducts (Hb-AGEs) from the hemoglobin-A_1C (HbA_1C) fraction. Precast polyacrylamide gels (Ampholine^® PAGplate) were used in Pharmacia LKB Multiphor II for this purpose. The separated bands for Hb-AGE and HbA_1C based on their isoelectric point (pI), were confirmed with the purifed fractions obtained from the cation exchange chromatographic technique. From the calibration curve, the pI values were found to be 6.748 and 6.495 for HbA_1C and Hb-AGE, respectively. The lowering of pI values for glycated hemoglobin, when compared to unglycated hemoglobin (pI = 6.852), can be attributed to the glycation at the amino terminals of the peptide chains. Increased reduction in pI value for Hb-AGE can be attributed to the effect of glycation of amino groups at various sites on the peptide chains, apart from the terminal amino groups. Fluorescence analysis was carried out for the purified fraction of Hb-AGE which showed the formation of a new fluorophor adduct having the excitation and emission maxima at 308 nm and 345 nm, respectively. Time-dependent formation of Hb-AGE under in vitro conditions was monitored by fluorescence (308/345 nm) over a period of 120 days, which showed its formation only after 3 weeks of incubation.