PROPERTIES OF A RECOMBINANT HUMAN HEMOGLOBIN WITH ASPARTIC-ACID 99(BETA), AN IMPORTANT INTERSUBUNIT CONTACT SITE, SUBSTITUTED BY LYSINE

Site-directed mutagenesis of an important subunit contact site, Asp-99 (beta), by a Lys residue (D99K(beta)) was proven by sequencing the ent ire beta-globin gene and the mutant tryptic peptide. Oxygen equilibriu m curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated tha t it had an increased oxygen affinity and a lowered but significant am ount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only margi nally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regu lators was limited by the mutant Hb. At concentrations of 0.2 mM or lo wer in heme, the mutant D99K(beta) Hb was predominantly a dimer as dem onstrated by gel filtration, haptoglobin binding, fluorescence quenchi ng, and light scattering. The purified dimeric recombinant Hb mutant e xists in 2 forms that are separable on isoelectric focusing by about 0 .1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the sa me masses for their heme and globin moieties as determined by mass spe ctrometry. The elution positions of the alpha- and beta-globin subunit s on HPLC were identical. Circular dichroism studies showed that one f orm of the mutant Hb had a negative ellipticity at 410 nm and the othe r had positive ellipticity at this wavelength. The findings suggest th at the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.