A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea).
Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.
Singh
Ritu
R
Department of Biosciences, Jamia Millia Islamia, New Delhi-110 025, India.
Ali Dar
Tanveer
T
Ahmad
Shandar
S
Moosavi-Movahedi
Ali Akbar
AA
Ahmad
Faizan
F
D002152:Calorimetry, Differential Scanning; D019791:Guanidine / Q000737:chemistry*; D006358:Hot Temperature; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D014508:Urea / Q000737:chemistry*
D011506:Proteins; D014508:Urea; D019791:Guanidine