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The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site- directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.

Potentiometric and conductometric studies of the nickel-thiomalic acid system, in aqueous medium of 0. lM KNO3, reveal the formation of two complexes; one light violet 1 : 1 predominating at pH 6.5-7.5 and another deep violet 1 : 2 in the pH range 8.5-10.0. The stability constants of the complexes formed have been determined by applying Calvin and Melchior's extension of Bjerrum's method at three different temperatures and were further refined by using alternative methods. The logK values (final) for 1 : 1 and 1 : 2 complexes at 20, 25, and 30 have been found to be 7.86, 7.87, 7.96, and 6.24, 6.31, 6.39 respectively. The values of the overall changes in ΔG, ΔH, and ΔS accompanying the reaction have also been evaluated at 25 and found to be -19.31 kcal/mole, -8.77 kcal/mole, and +35.36 cal/deg respectively.