MICROCALORIMETRIC CHARACTERIZATION OF THE ANION-EXCHANGE ADSORPTION OF RECOMBINANT CYTOCHROME B(5) AND ITS SURFACE-CHARGE MUTANTS

The adsorption of recombinant soluble tryptic fragment of rat cytochro me b(5) on the strong anion exchanger Mono Q was studied using isother mal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed prot ein show increasingly endothermic (unfavorable) enthalpies of binding with increasing surface coverage, confirming the heterogeneous nature of binding. The enthalpy of adsorption declines toward zero at higher loadings. At low surface coverage, enthalpies increase linearly with t emperature, giving rise to a positive value of Delta C-p. Enthalpies o f adsorption depend strongly on the history of the adsorbent. DSC is u sed to show that cytochrome b(5) is stable in both free and adsorbed s tates at all temperatures used in the titration calorimetric experimen ts. Site-directed mutants of recombinant cytochrome b(5) carrying sing le charge-neutralizing substitutions are used to test the contribution s of particular residues to the thermodynamics of adsorption. Like tho se derived from van't Hoff analysis of equilibrium adsorption isotherm s and HPLC retention data, calorimetric enthalpies of adsorption are p ositive, confirming the dominant role of entropic effects in ion-excha nge adsorption in this system.