I. Kinetic Evaluation of Hemoglobin Assembly from Normal and Variant Heme Subunits. The reconstitution of oxyhemoglobin from its oxygenated heme subunits involve three reactions. Dissociation of oligomeric subunits into monomers (Reaction 1) must occur before they can combine to form AlphaBeta dimers (Reaction 2). Subsequently, two dimers aggregate to form the Alpha2Beta2 hemoglobin tetramer (Reaction 3). The rate of Alpha and Beta monomer association (Reaction 2) will be determined over a wide range of protein concentration, pH and ionic strength conditions by direct rapid mixing experiments in a stopped-flow device. This reaction will be monitored by both absorption and fluorescence spectrophotometry since these spectral properties of intact hemoglobin differ from those of its isolated Alpha and Beta subunits. Several normal and variant human hemoglobins will be studied to test a proposed electrostatic model of subunit assembly. The rate of dissociation of liganded Beta chain tetramer (Reaction 1), a known rate limiting step in hemoglobin assembly, as well as the rate of dimer aggregation (3) will also be determined for selective variants using spectroscopic techniques. These experiments should provide new insights into the role of subunit assembly as a post-synthetic determinant of the distribution of hemoglobin in the erythrocyte of normal individuals and those with various hematological disorders, especially the thalassemias. II. Kinetic Investigation of the Role of Globin Intermediates in the Assembly Process. During the hemoglobin assembly process in vivo the heme moiety must be inserted into the newly synthesized polypeptide chain. Globin and heme intermediates could be involved in the assembly of the hemoglobin tetramer. The reaction of ferroprotoporphyrin IX with hemoglobin globin (Alpha0; Beta0), with semi-hemoglobins (Alpha 2 heme Beta-0-2 and Alpha 02 Beta 2 heme) and with chain globins (Alpha-0; Beta-0) will be monitored using rapid kinetic techniques. Both ultraviolet and visible spectroscopic methods will be employed to measure the rate of combination of heme and globin subunits. Detailed information on the kinetics of heme insertion, as well as, the rates of combination of possible globin and heme subunit intermediates is essential to defining the pathway by which nascent polypeptide chains are transformed into biologically active hemoglobin oligomers in vivo.
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