Gel chromatographic analysis of ficin under native and under denaturing conditions

The hydrodynamic behaviour of a commercial ficin preparation was studied by analytical gel chromatography on Sephacryl S-200 HR column under native and under denaturing (in presence of 9 M urea / 6 M guanidine hydrochloride, GdnHCl) conditions. The commercial ficin preparation was fractionated into seven distinct active components (I-VII) under native conditions. The elution of the components (I-III) as compared to alpha-chymotrypsinogen and the elution of the components (IV-VII) at a volume exceeding the total bed volume were suggestive of the interaction between the protein molecules and the gel. Treatment of ficin with denaturants (9 M urea / 6 M GdnHCl) resulted in its elution in the form of two peaks, indicating the presence of two classes of conformers differing in their stability towards denaturants. Analysis of the chromatographic data yielded the available Stokes radii of ficin (peak I fraction) as 9.2 angstrom (under native conditions), 25.8 angstrom (in presence of 9 M urea) and 40.9 angstrom (in presence of 6 M GdnHCl). A comparison of Stokes radii of ficin obtained under denaturing conditions suggested complete denaturation of ficin in 6 M GdnHCl compared to 9 M urea which produced significantly less conformational alteration.