INFLUENCE OF AA SUBSTITUTIONS ON STABILITY OF TETRAMER-DIMER INTERFACE OF HBS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An ongoing and productive collaboration is still in progress between Prof. Manning's laboratory at Northeastern University and ours. The experimental goal of this collaborative endeavor is at the complete characterization by mass spectrometry of either naturally occurring or genetically engineered mutations in the polypeptide chains of human hemoglobins. Recently, our laboratories began a project with Prof. J. Eric Russell's laboratory to study embryonic hemoglobins produced in transgenic mice. Our studies aim at a better understanding of the long-range communications within hemoglobin tetramers which affect the efficiency of the oxygen transport to peripheral tissues. We are currently investigating the relationship of certain amino acid residues near the tetramer-dimer interface of human hemoglobin. Mass spectrometry is being used to characterize the constituting polypeptide chains in hemoglobin using a three-step approach that consists in (1) analyzing the intact mass of the polypeptide chains by electrospray ionization mass spectrometry; (2) digesting the polypeptide chains with a specific proteinase and obtaining peptide maps by MALDI-TOF/MS; and (3) sequencing the peptides containing the mutations by mass spectrometric fragmentation on an electrospray ionization ion trap mass spectrometer.