Allosteric sensitivity in hemoglobin at the alpha-subunit N-terminus studied by hydrogen exchange.
Allosteric structure change in human hemoglobin was studied by hydrogen-tritium-exchange methods. The functional labeling method used takes advantage of the change in H-exchange rate at allosterically involved sites to selectively label, with tritium, H-exchange sites that are fast in one protein state and slow in another. The position of the labeled sites can then be located by the medium-resolution fragmentation-separation method. These methods reveal 5 allosterically sensitive, H-bonded, peptide NH's within the first 12 residues of the alpha chain. All five exchange with solvent protons at similar rates in deoxyhemoglobin (T form), and all shift to a new rate, about 30-fold faster, in the liganded protein (R) form. This indicates a decrease in structural stability at the alpha-chain N-terminus in going from the T to the R form, consistent with the loss of stabilizing interactions in that segment. The results indicate a loss of perhaps 2 kcal/mol in stabilization free energy and thus document a significant role for changes at the alpha-chain N-terminus in the allosteric transition.
D000494:Allosteric Regulation; D000596:Amino Acids / Q000032:analysis; D016282:Aspartic Acid Endopeptidases; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011865:Radioisotope Dilution Technique; D014316:Tritium
D000596:Amino Acids; D006454:Hemoglobins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014316:Tritium; D006859:Hydrogen; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases