Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

Abstract

Site-directed mutagenesis of an important subunit contact site, , by a Lys residue (D99K(@)) was proven by sequencing the entire @-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally 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 regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(@) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists 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 same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the a-and @-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(@) recombinant mutant forms have differences in their heme-protein environments.

A Recombinant Sickle Hemoglobin Triple Mutant with Independent Inhibitory Effects on Polymerization

“…Its oxygen affinity, cooperativity, response to negatively charged effectors, alkaline Bohr effect, and the tetramer/dimer dissociation constant were the same as those for HbS. These results, together with extensive characterization of recombinant hemoglobins by a variety of biochemical criteria (15,25,26,29), are consistent with the expression by the yeast system of a native hemoglobin molecule with the correct N-terminal processing. Thus, we have no evidence for any misfolding of the triple mutant as reported for other recombinant hemoglobins made using E. coli as a production host (30).…”

“…With an acetonitrile gradient from 40 to 45%, the ␣-and ␤-chains did not separate due to the combined effects of the slightly increased elution of the ␤-chain having the L88A(␤) mutation (6) and the considerably decreased elution of the ␤-chain with the K95I(␤) mutation (12). The chains were suc- Peptide Mapping-Tryptic peptide mapping of the isolated ␤-chain was performed as described previously (6,15,25,26) to verify the expected mutations. The peptides were separated on a Vydac C-18 column using a shallow gradient of acetonitrile (from 12 to 44% in 50 min) in 0.05% HCl (Fig.…”

“…Analytical Methods-Isoelectric focusing, amino acid analysis, and other procedures were performed as described earlier (12,15,25). To isolate the ␣-and ␤-globin chains, a Vydac C-4 column was equilibrated with acetonitrile in 0.1% trifluoroacetic acid and eluted as described under "Results."…”

“…To isolate the ␣-and ␤-globin chains, a Vydac C-4 column was equilibrated with acetonitrile in 0.1% trifluoroacetic acid and eluted as described under "Results." The isolated ␤-globin chains were digested with trypsin, and the resulting peptides were separated on a Vydac C-18 reversed phase column using an acetonitrile gradient in 0.05% HCl, a modification from previous studies (12,15,25).…”

Normal and Abnormal Protein Subunit Interactions in Hemoglobins

“…the natural variant Hb Rothschild (8) and the recombinant hemoglobin D99K (␤) (Asp-99 (␤) 3 Lys) (9), which have disruptive amino acid substitutions preventing formation of the tetramer-dimer interface and thus the central cavity. However, Hb from some clams is a functional dimer (10,11), and it has a cooperative mechanism that is completely different from that found in human hemoglobin because its monomers fit together differently than in vertebrate hemoglobins.…”