Optical Rotatory Dispersion and Circular Dichroism of Human Carbonic Anhydrases B and C

Abstract

Abstract Studies of optical rotatory dispersion (ORD) and circular dichroism (CD) are reported on human carbonic anhydrases B and C in the spectral region below 320 mµ. Most of the observed CD spectrum of each enzyme could be described in terms of three principal Gaussian bands: a strong negative band near 216 mµ, a weaker negative band near 270 to 275 mµ, and a positive band intermediate between the other two in position. Above 260 mµ, all CD values are negative; there is clear evidence of fine structure above 280 mµ, involving at least two additional CD bands. The ORD spectra above 260 mµ show several peaks and troughs, which are characteristic for each enzyme. The principal troughs at shorter wave lengths lie at 222 mµ for Enzyme B and at 226 mµ for Enzyme C. At still shorter wave lengths, the ORD values rise to low peaks, at 204 mµ, with [m'] = -300 for Enzyme B and +2750 for Enzyme C. These patterns are very different from those characteristic of either α-helical or β structures. Asymmetrical interactions involving the aromatic side chains almost certainly make important contributions to ORD and CD at the shorter wave lengths, as they clearly do at the longer wave lengths, above 250 mµ. On acid denaturation, the longer wave length Cotton effects vanish, and the ORD and CD spectra alter in such a way as to suggest the presence of 10 to 20% α-helix in the acid-denatured proteins. The changes on exposure to high pH are more complex. Near pH 11, the negative CD band near 270 to 275 mµ becomes less intense and shifts to longer wave lengths; the positive band at 232 mµ in Enzyme B also shifts to longer wave lengths. It is tentatively suggested that these bands arise in part from interactions of tyrosine residues. The fine structure pattern between 280 and 310 mµ undergoes changes, but in Enzyme B it still persists even at pH 13. Interactions involving tryptophan may be involved. The positive CD band at 249 mµ in Enzyme C disappears at pH 11.5, and the ORD spectrum of this enzyme also indicates more drastic structural alterations at high pH than those found in Enzyme B. The ORD spectra of both enzymes at pH near 13 show two troughs, one near 231, the other near 210 mµ, the former being deeper for Enzyme B, the latter for Enzyme C. The CD bands of Enzyme B are still observed in 2 m guanidine-HCl, although the long wave ultraviolet bands are weaker than in the native protein; in 4 m guanidine-HCl they are abolished. ORD studies of the two enzymes in 1 m guanidine-HCl indicate that the Cotton effects due to the aromatic absorption bands disappear rapidly in this solvent in solutions of Enzyme C, whereas Enzyme B is much more stable. ORD spectra have been calculated from the CD data for the native enzymes by the use of a Kronig-Kramers transform with a computer program. Comparison with the observed ORD data shows good general agreement with the pattern of the observed troughs and peaks, especially for Enzyme C. The computed spectra are displaced to more positive [m'] values than the observed spectra, indicating that there must be large negative contributions to the circular dichroism at wave lengths below 200 mµ.