Denatured States of Ficin Induced by Urea and Guanidine Hydrochloride

Objective: To compare states of ficin denatured by 9 M urea and 6 M guanidine hydrochloride since a few proteins have shown different unfolded states upon denaturation with these agents. Methods: Intrinsic fluorescence, tryptophan fluorescence, far-UV spectral signal and enzyme activity were used to characterize native and different denatured states of ficin. Results: Treatment produced different denatured states when probed by intrinsic and tryptophan fluorescence. Both states differ significantly in magnitude of change in fluorescence intensity and extent of red shift. Red shifts of 12 and 17 nm were observed in the emission maximum with guanidine hydrochloride, and of 3 and 10 nm with urea using intrinsic and tryptophan fluorescence respectively. There were significant differences in magnitude of hypochromism and extent of red shift of far-UV absorption spectra. Guanidine hydrochloride produced total loss and urea 65% loss of enzyme activity. Conclusion: Denatured states of ficin produced by urea and guanidine hydrochloride were significantly different and guanidine hydrochloride was more effective than urea. These results may be useful in determining the structure-activity relationship of ficin.