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Nonideality and protein thermal denaturation
Author(s) -
Waldner Jennifer C.,
Lahr Steven J.,
Edgell Marshall Hall,
Pielak Gary J.
Publication year - 1999
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(199905)49:6<471::aid-bip5>3.0.co;2-z
Subject(s) - chemistry , differential scanning calorimetry , enthalpy , denaturation (fissile materials) , virial coefficient , thermodynamics , calorimetry , protein aggregation , analytical ultracentrifugation , ultracentrifuge , chromatography , biochemistry , nuclear chemistry , physics
We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two‐state model. At concentrations greater than several hundred μ M , however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two‐state model. © 1999 John Wiley & Sons, Inc. Biopoly 49: 471–479, 1999