Graduate Thesis Or Dissertation
 

Allosteric properties and the association equilibria of hemocyanin from Callianassa californiensis

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  • Monomer-tetramer association equilibrium and the oxygen binding of the hemocyanin from Callianassa californiensis were measured at a number of pH values and magnesium ion concentrations. Magnesium binding of the hemocyanin was also measured at several pH values. A thermodynamic model, which satisfied the constraints from the experimental results, was developed and the parameters in the model were determined from the experimental data. For the measurement of association equilibrium, weight average sedimentation coefficients were first measured by velocity sedimentation at a number of pH values and Mg²⁺ concentrations. It turned out that at pH values above 7.65 the reequilibration process was so slow that the boundaries of both components were clearly separated and the plateau region between the two boundaries was virtually horizontal. The ratio of the components from the velocity sedimentation was thus confirmed to be the equilibrium value under the conditions. At pH 8.0 the apparent equilibrium constant was determined for both oxy and deoxy state. The association profile with respect to the magnesium ion concentration shifted significantly to the right upon deoxygenation, indicating stronger association for the oxygenated form. Oxygen binding studies revealed that the role of Mg²⁺ and H⁺ can be interpreted as allosteric effectors in the framework of the "extended non-exclusive" Monod-Wyman- Changeux theory. It was also shown that the hemocyanin could show a high cooperativity of oxygen binding (as judged from the Hill coefficient, nH), even when no significant amount of tetramer is present, e.g. at pH 8.2 with 10 mM MgC₁₂. This supported the suggestion by Miller and Van Holde (1974) that the monomer, which consists of six polypeptide chains is the allosteric unit. Altogether, about 42 strong magnesium binding sites were found per monomer (17S) from the Mg²⁺ binding study. Competition between one Mg²⁺ and two H⁺ was suggested. No significant difference in Mg²⁺ binding between oxy and deoxy states was found, which implied that the number of the oxygen-linked Mg²⁺ binding sites was small compared to the total number of the Mg²⁺ binding sites. The data analysis based on the model which is developed herein further revealed that: (i) among the 42 magnesium binding sites about four were oxygen linked and about three were involved in the association process; and (ii) the ratio of the allosteric conformational equilibrium constants in monomer and tetramer has the relatively small value of 1.7. The latter observation implies that the shift of the association equilibrium upon oxygenation will not affect the oxygen binding curve significantly. This observation justifies the analysis of the oxygen binding curve, where the effect of the shift in association equilibrium is neglected. A possible relationship between the thermodynamic model and the structural one based on the recent x-ray diffraction study by Kuiper et al. (1975) will also be discussed.
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file details ArisakaFumio1977.pdf 2017-08-07 Pubblico Scaricare