Comparison of the dynamic structure of alpha-chymotrypsin in aqueous solution and in reversed micelles by fluorescent active-site probing.

Steady-state fluorescence measurements of the progressive red-shift of the center of gravity of the emission band as function of degree of hydration, w,, defined as [H,O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at w, > 12. Timeresolved fluorescence measurements of Ant-CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < w,< 5) can be observed, which correlate with enzyme activity measurements in the same w, region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational-correlation time of the enzyme in AOT reversed micelles seems to be independent on w, (5 < w, < 29), which suggests that the enzyme creates its own micelle.