TY - JOUR
T1 - Microcalorimetric characterization of the anion-exchange adsorption of recombinant cytochrome b5 and its surface-charge mutants
AU - Gill, Davinder S.
AU - Roush, David J.
AU - Shick, Kari A.
AU - Willson, Richard C.
N1 - Funding Information:
We would like to thank Dr. John Brandts, Sam Williston, and the late Tom Wiseman of MicroCal, Inc. for their indispensable help in designing the titration calorimetry experiments. We would also like to thank Dr. Jan-Christer Janson of Pharmacia LKB for the generous gift of Mono Q. E. coli strains producing cytochrome b 5 were the generous gift of Dr. Steve Sligar at the University of Illinois. Drs. Henry Pownall and Martha Mims of the Baylor College of Medicine, Houston, kindly allowed us use of their DSC equipment, and Dr. Barry McKeone provided valuable assistance in performing DSC experiments. The skillful assistance of Rene Ochoa, Colin Johnston, Henry Chang, Jerry Mulholland, and Don MacManus is gratefully acknowledged. Funding for this project was provided by NSF through CTS-8910087 and through a PYI award to R.C.W., and by the Welch Foundation.
PY - 1995/10/27
Y1 - 1995/10/27
N2 - The adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the strong anion exchanger Mono Q was studied using isothermal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed protein 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 temperature, giving rise to a positive value of ΔCp. Enthalpies of adsorption depend strongly on the history of the adsorbent. DSC is used to show that cytochrome b5 is stable in both free and adsorbed states at all temperatures used in the titration calorimetric experiments. Site-directed mutants of recombinant cytochrome b5 carrying single charge-neutralaizing substitutions are used to test the contributions of particular residues to the thermodynamics of adsorption. Like those derived from van 't Hoff analysis of equilibrium adsorption isotherms and HPLC retention data, calorimetric enthalpies of adsorption are positive, confirming the dominant role of entropic effects in ion-exchange adsorption in this system.
AB - The adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the strong anion exchanger Mono Q was studied using isothermal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed protein 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 temperature, giving rise to a positive value of ΔCp. Enthalpies of adsorption depend strongly on the history of the adsorbent. DSC is used to show that cytochrome b5 is stable in both free and adsorbed states at all temperatures used in the titration calorimetric experiments. Site-directed mutants of recombinant cytochrome b5 carrying single charge-neutralaizing substitutions are used to test the contributions of particular residues to the thermodynamics of adsorption. Like those derived from van 't Hoff analysis of equilibrium adsorption isotherms and HPLC retention data, calorimetric enthalpies of adsorption are positive, confirming the dominant role of entropic effects in ion-exchange adsorption in this system.
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U2 - 10.1016/0021-9673(95)00393-2
DO - 10.1016/0021-9673(95)00393-2
M3 - Article
C2 - 8520668
AN - SCOPUS:0028793481
SN - 0021-9673
VL - 715
SP - 81
EP - 93
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 1
ER -