In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process

In-situ confocal Raman spectroscopy combined with relative humidity (RH) control technique was used to study the sequential dehydration process of insulin crystals. By gradually decreasing the ambient RH of the insulin crystal, the content of the hydration water in the crystal was quantitatively controlled. Tyrosine (Tyr) residues were very sensitive to the micro-environmental changes, and four Raman features 828 cm^-1, 852 cm^-1, 1174 cm^-1 and 1206 cm^-1 of Tyr were employed to monitor the dehydration process. Taking advantage of the ratios I₈₅₂/I₈₂₈ at different RH values, the mole fractions of the 'exposed' and 'buried' Tyr residues were estimated. Moreover, using the ratio I₁₁₇₄/I₁₂₀₆ as an indicator of the dehydration process, three RH regions were discriminated. This is believed to imply that different types of the hydration water were lost step by step, i.e. firstly the 'second-layer' and 'first-layer' classes, then the 'contact' class, and finally, the 'inside' class. In addition, the profile of the amide I band was observed to gradually change with RH. By band fitting of the amide I region, changes in secondary structure were quantitatively determined. And the results showed that nearly 17% of α-helix converted into β-sheet with RH decreasing from 92% to 2%.