Activity and mobility of subtilisin in low water organic media: hydration is more important than solvent dielectric.

The relationship between hydration, catalytic activity and protein dynamics was investigated for subtilisin Carlsberg in organic solvents with low water content. The organic media were cyclohexane, dichloromethane or acetonitrile, with controlled thermodynamic water activity (aw). Catalytic rate profiles showed the same dependence on aw for the three different solvents. The structural mobility of the enzyme in air and organic media was probed by proton solid-state NMR relaxation measurements. Both spin-lattice relaxation time (T1 ) and line width at half height (apparent spin-spin relaxation time (T2)) were determined for protein which was exchanged and hydrated with D2O. We found NMR relaxation was much more dependent on aw than medium identity (despite very different dielectrics) showing that enzyme hydration is the primary determinant of mobility. Results suggest that initial hydration up to aw 0.22 causes rigidification of part of the protein structure. As aw is increased further, enzyme mobility is found to increase. Above aw 0.44, a large increase in the proportion of more mobile protons coincides with a steep rise in catalytic activity for the enzyme in each of the solvents studied.

The metabolism of fluorometholone by bovine cornea.

Isolated bovine cornea was found to reduce the 20 ketone group in fluorometholone (FML) to produce a mixture of 20 alpha- and 20 beta-dihydrofluorometholone (DHFML). Incubation of FML with bovine cornea at pH 7.4 in phosphate buffer produced an increase in the amount of reduction products up to 8 h; after this time the rate of reduction decreased. The reduced products were quantified by GC-MS against [2H3] 20 alpha-DHFML. Variable amounts of 20 beta-DHFML were detected in six samples of human aqueous humour following topical application of 50 micrograms of FML to the eye. The rapid metabolism of the side chain of FML may account for the unexpected low concentrations of FML which are found in human aqueous humour following its application to the surface of the eye.