A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

Abstract

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca2+ and is eluted by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca2+-dependent affinity chromatography on W-7 Sepharose. The Ca2+-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca2+-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca2+ in the aromatic and far-uv range. In the presence or absence of Ca2+, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that alpha-helical contents of both S-100a and S-100b decreased with addition of Ca2+. Ca2+-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 microM Ca2+; on the other hand, S-100a required concentrations above 8 microM. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca2+ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca2+ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.