The long-term objective of this research is to study the functional properties of abnormal human hemoglobins by measuring their oxygen binding equilibria under various controlled conditions of pH, organic phosphates, and other ion conditions. The results will be used for two purposes. First, attempts will be made to correlate functional properties with structural alterations in order to better understanding of contributions of specific amino acid residues in hemoglobin to its oxygen transport function. Second, attempts will be made to explain the presence or lack of clinical abnormalities such as erythrocytosis or anemia with specific abnormal hemoglobins with altered function.
The specific aims i nclude: 1) Refine and improve procedures and systems for isolating electrophoretically silent abnormal hemoglobins and for measuring oxygen-hemoglobin in the equilibrium reaction. 2) Screen previously unstudied and all new abnormal hemoglobins as they become available for general parameters of O2 binding (P50, cooperativity, Bohr effect and allosteric effects of anions) in order to identify whether or not the altered residue influences normal oxygen binding. 3) Examine in detail any abnormal hemoglobin found to have altered oxygen binding properties using an automatic recording system which is capable of estimating the four Adair constants for the oxygenation reaction in order to determine as precisely as possible which part of the over-all reaction of oxygen and hemoglobin has been altered by the mutational change. 4) Correlate changes in oxygen equilibrium reactions with altered protein structure in order to better understand the normal reaction mechanism, and 5) evaluate to what extent changes in oxygen binding properties of mutant hemoglobins can be correlated with other hematological measurements such as erythrocyte 2,3 DPG, hematocrit, and hemoglobin concentration. The principal method used will be the measurement of oxygen equilibrium curves by the automatic recording system of Imai which measures simultaneously the functional saturation of hemoglobin and oxygen pressure over a range of less than 1.0% saturation to over 99.9% saturation in the case of normal hemoglobin A. On-line microcomputer storage of data and subsequent computation of the results according to the Adair analysis and other reaction models will be employed for interpretation.
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