FLUORESCENCE SPECTROSCOPIC STUDY OF SUBTILISINS AS RELEVANT TO THEIR CATALYTIC ACTIVITY IN AQUEOUS-ORGANIC MEDIA

The fluorescence spectra of subtilisin Carlsberg (STC) and subtilisin BPN' (STB) were measured in aqueous-organic mixed solvents over a wide range of solvent composition. The tryptophan fluorophores were used a s probes of the environmental polarity, and the structural changes of the enzymes were detected as changes of the difference in the emission wavelength between the enzymes and N-acetyl-L-tryptophan ethyl ester (DELTAlambda(em)). It was found that DELTAlambda(em) was well correlat ed with the catalytic activity of the enzymes, as expressed by the hyd rolysis rate of N-acetyl-L-tyrosine ethyl ester. For example, the char acteristic dependency of the activity of STC on the solvent compositio n in acetonitrile-water was closely related to the change in DELTAlamb da(em). Similar correlations between DELTAlambda(em) and the catalytic activity were observed for subtilisins in aqueous 1,4-dioxane, THF, D MF, DMA, and DMSO solutions with high correlation coefficients, except for STB in water-DMF and -DMA. The results are discussed in terms of the structural modification of the enzymes in these aqueous-organic me dia.