Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry.
Pressure perturbation calorimetry is a new technique that measures the heat change in a solution that results when the pressure above the solution is changed. When used in a differential calorimeter containing a dilute solution of solute in the sample cell and the corresponding buffer in the reference cell, the measured differential heat can be used to calculate the thermal coefficient of expansion of the partial volume of the solute, alpha. For proteins in dilute aqueous solution, alpha is dominated by a temperature-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to alpha can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, ribonuclease A, and T4 lysozyme) were examined carefully by this technique, allowing estimates of various volumetric parameters including the volume change resulting from thermal unfolding of each protein. For ribonuclease A, results obtained in both water and deuterium oxide led to an estimate of the accessible surface area of the native protein of approximately 45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to ribonuclease A led to changes in alpha, suggesting a burial of some surface area in the ligand-protein complex.
Lin
Lung-Nan
LN
MicroCal, LLC, 20 Industrial Drive East, Northampton, Massachusetts 01060, USA.
Brandts
John F
JF
Brandts
J Michael
JM
Plotnikov
Valerian
V
D000818:Animals; D002151:Calorimetry / Q000379:methods*; D017666:Deuterium Oxide; D019791:Guanidine; D006801:Humans; D006874:Hydrostatic Pressure; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013816:Thermodynamics; D014867:Water
D011506:Proteins; D014867:Water; D012259:Ribonuclease, Pancreatic; D017666:Deuterium Oxide; D019791:Guanidine