ANION-EXCHANGE CHROMATOGRAPHIC BEHAVIOR OF RECOMBINANT RAT CYTOCHROMEB(5) - THERMODYNAMIC DRIVING FORCES AND TEMPERATURE-DEPENDENCE OF THESTOICHIOMETRIC DISPLACEMENT PARAMETER-Z

The HPLC anion-exchange isocratic retention behavior of the recombinan t soluble core of wild type rat cytochrome b(5) on Mono Q HR 5/5 was i nvestigated as a function of temperature and sodium chloride concentra tion at fixed eluent flow-rates. Retention was measured over a range o f eluent flow-rates at a specified temperature to determine if true ad sorption equilibrium could be approximated by the HPLC method. Apparen t Van't Hoff enthalpies of adsorption obtained from the HPLC retention data were positive, indicating an entropically driven spontaneous ads orption process, and were found to decline with increasing ionic stren gth. The retention results were interpreted in terms of the stoichiome tric displacement model to obtain the apparent number of binding sites in the contact region, Z, as a function of temperature and of protein concentration. Z was found to depend significantly on temperature, ev en under conditions of nearly complete protein recovery, but did not d epend on protein concentration at the low loadings studied.