Solvent isotope effect on the differences in structure and stability between normal and deuterated proteins

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔHd) and the temperature (td) of thermal denaturation of normal (nondeuterated) (H‐PC) and deuterated (D‐PC) phycocyanins in D2O solvent. Values of td in D‐PC are about 5–7°C lower than those in H‐PC. The magnitudes of ΔHd in D‐PC are only 21–32% of those in H‐PC. During the protein unfolding, the heat‐capacity changes (ΔCp) in D‐PC are also lower than those in H‐PC. CD was employed to evaluate the secondary structure and the urea denaturation of these proteins in D2O solvent. These proteins have about the same α‐helix content. D‐PC is less resistant to the denaturant urea than is H‐PC. In general, the apparent free‐energy change in the process of protein unfolding at zero denaturant concentration is higher in H‐PC than in D‐PC. Comparisons of the present results for D2O solvent with those previously reported for H2O reveal that solvent isotope effect essentially does not change the α‐helix content in H‐PC and D‐PC. However, D‐PC or H‐PC has a higher random‐coil content in its secondary structure in D2O than in H2O. Substitution of H2O with D2O as the solvent increases td in both D‐PC and H‐PC, lowers ΔHd in H‐PC, and greatly lowers ΔHd in D‐PC. The deuterium solvent isotope effect does not change ΔCp in H‐PC but lowers ΔCp in D‐PC. In the urea denaturation, the magnitudes of (Cu)1/2 in H‐PC and D‐PC are not affected by such a solvent effect, whereas those of ΔG are greatly increased. These results are correlated with the structure and stability of the proteins.