The observations of the presence of entropy driven intermediate steps of oxygénation in various hemoglobin systems from different species and from different chemical treatments [Bucci, E. et al. (1991) Biochemistry 30; 3195; (1993) Biochemistry 32; 3519; Biophys. J. in press; Johnson et al. (1992) Biochemistry 31: 10074; (1995) Biophys. Chem. 59; 107] pointed out the necessity of high precision measurements of oxygen binding in order to understand the role of enthalpic/entropic effects in the allosteric transition. We have simulated the effects of different experimental and analytical approaches to the accuracy of recovered parameters. Using determination of oxygen binding capacity [Dicera and Gill (198) Biophys.Chem.32:149], eliminates some optical parameters from the data improving the accuracy of the analysis. Also, we designed a global approach where five or more binding capacity curves measured at different temperatures are simultaneously analyzed. This restricts the confidence limits of the recovered values and corrects distortions due to the bias of the experimental noise. We conclude that only global analysis of multi-temperature measurements can recover precise values of the Adair's binding constants and their enthalpies. Temperature is an important factor which strongly affects the distribution of the intermediates species of oxygénation. Our results show that temperature critically affect the apparent T/R equilibrium. This implies that interpretation of modifications of oxygen affinity based on single temperature isotherms may be temperature dependent and misleading.
|State||Published - 1 Dec 1998|