PREPARATION, PROPERTIES, AND PLASMA RETENTION OF HUMAN HEMOGLOBIN DERIVATIVES - COMPARISON OF UNCROSSLINKED CARBOXYMETHYLATED HEMOGLOBIN WITH CROSS-LINKED TETRAMERIC HEMOGLOBIN

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of almost-equal-to 70%. The predominant product was crosslinked between subunits within a tetramer and had a M(r) of 64,000; no higher M(r) species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha-chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.