Initial hydration steps in lipase studied by means of water sorption isotherms, FTIR spectroscopy and thermally stimulated depolarization currents

The role of water in lipases, a class of proteins endowed with a large external hydrophobic surface, is not yet fully understood. To analyse the water-related structural properties and the possible implications for the protein functionality, three experimental techniques such as water sorption isotherms, thermally stimulated depolarization currents (TSDCs), and Fourier transform infrared spectroscopy were applied to pellets and/or films of lipase from Candida lipolytica, prepared at very low hydration degrees h, ranging between 0.003 and 0.457 g(water)/g(protein). Two main broad TSDC bands (a weak one peaking at T congruent to 160 K and a strong one at T congruent to 260 K) were detected. The peak amplitudes and positions critically depend not only on the water content, but also on the previous hydration history of the sample. FTIR spectra monitored: (1) the amplitude and position changes of the characteristic optical absorption bands (amide A, B, 1, and 11) as a function of the humidity level, (2) the presence of a considerable amount of P-sheet structure at high hydration degrees, and (3) a conformational transition induced by drastic dehydration treatments. Complementary sorption isotherms, performed by means of a gravimetric method, showed a marked hysteresis in the lipase-aqueous solvent interaction. The whole set of results provides a model for the initial steps of the lipase hydration kinetics. At h = 0.009, 13 water molecules are buried in the macromolecule, probably bound to the peptidic backbone. At h = 0.037 all polar and charged free groups are hydrated. At higher h a solution phase begins, and at h = 0.457 about 660 water molecules are accommodated around the protein, giving rise to three to four complete layer coverages.