Effects of ligand size on pH and organic phosphate-dependent affinity changes in carp hemoglobin as measured by isonitrile binding.
The equilibria of the binding of methyl and ethyl isonitrile to carp hemoglobin have been measured at three pH values in the presence and absence of inositol hexaphosphate. The binding of methyl isonitrile is characterized by a higher overall dissociation constant, C1/2, and a higher Hill coefficient, n, than that of the ethyl derivative. The former is consistent with the greater hydrophobicity of ethyl isonitrile, and the latter is probably due to a greater intrinsic difference or heterogeneity in the binding affinities of the alpha- and beta-chains for the larger ligand. Changes in log C1/2 which result from alterations in pH or addition of organic phosphate are the same for both ligands within experimental error. This result is not consistent with affinity changes being the result of steric interactions between the protein and the ligand. At pH 6 in the presence of inositol hexaphosphate, equilibrium parameters estimated from overall rates of ligand binding and dissociation are in good agreement with direct equilibrium measurements. This is consistent with the protein being in a low-affinity, T-like state even when saturated with ligand under these conditions, resulting in a loss of cooperativity in ligand binding. At high pH, ligand binding remains cooperative, as evidenced by n values greater than unity, a general lack of agreement between measured equilibrium parameters and those estimated from overall kinetic constants, and differences in the kinetics of ligand binding as observed by rapid-mixing and flash photolysis techniques. Thus, the deoxygenated state of carp hemoglobin at high pH does not appear to be a good model of a deoxygenated R quaternary structural state.
Lin
M J
MJ
Department of Biochemistry, State University of New York, Buffalo 14215.
Noble
R W
RW
Winterhalter
K H
KH
DiIorio
E E
EE
D000818:Animals; D002347:Carps; D006454:Hemoglobins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism; D011485:Protein Binding
D006454:Hemoglobins; D008024:Ligands; D009570:Nitriles; C008462:methylisonitrile; C001187:ethyl isocyanide; D010833:Phytic Acid