This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the {alpha}- or the {beta}-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the {alpha}-subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer?dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N-terminals of the {beta}-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer?dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the {alpha}-subunit, the slope of the plot of the tetramer?dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the {alpha}-subunits is less extensive than that of the {beta}-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.
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