Thermodynamics of ligand binding and allosteric transition in hemoglobins. Reaction of Hb trout IV with CO.

The reaction of hemoglobin trout IV with carbon monoxide has been studied in three parallel sets of experiments comprising: (1) microcalorimetric measurements in different buffers at two pH values (7.1 and 8.5) and two temperatures (20 °C and 5 °C) to determine the overall value of the enthalpy change of ligand binding as well as its point values as a function of fractional saturation with CO; (2) a study of the CO binding curves at three pH values (from 5.7 to 7.4) and at 23 °C and 5 °C in order to explore the pH and temperature dependence of the model-independent binding parameters which describe the system; (3) differential titrations in 0.2 m-sodium chloride at 23 °C and 5 °C of the deoxy and CO saturated forms of the molecule in order to determine the overall CO Bohr effect as a function of temperature. Analysis of the results of these three different sets of experiments at a purely phenomenological level shows that they are in satisfactory agreement. Further analysis in terms of a modified two-state allosteric model shows how the three model parameters k_T, k_R, and L vary with pH and temperature. From this it appears that the essential feature of the functional behavior of trout IV hemoglobin, which is characteristic of hemoglobins of teleost fish in general, lies in a proton-induced shift in the allosteric equilibrium constant. The data indicate, semiquantitatively, that the apparent enthalpy change corresponding to the allosteric equilibrium constant is pH-dependent and positive.