A high-precision gas-solution microcalorimeter together with the Gill thin-layer optical apparatus and isobaric temperature scan technique are used to study the binding reaction of oxygen with different hemoglobin systems. Independent optical and calorimetric experiments on human, bovine and deer hemoglobins reveal that the enthalpies of ligation at subsequent steps of oxygenation are not constant, indicating the presence of enthalpy and entropy driven binding steps in alternating fashion. These phenomena should be referred to conformational changes of the protein as well as to changes of the interaction of the protein surface with the solvent. To study the effect of protein conformational changes we extend our study of natural human and bovine hemoglobin to its crosslinked derivatives obtained by bridging the b subunits of the tetramer between the b82 lysines across the b cleft. Linkers of different length (4, 6 and 10 carbon) which stabilize tetrameric structure are used. Variation of the length of the linkers differently affects the conformational flexibility of tetrameric hemoglobins. In order to investigate solvent effects we propose to study oxygen binding of various hemoglobin systems in the presence of osmolytes which change the activity of water. In order to increase the accuracy of experimental results we are developing a software for global analysis of calorimetric and optical data of oxygen binding.
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