Abstract
Abstract The optical rotatory dispersion of bovine carbonic anhydrase B and human carbonic anhydrase B and C is investigated between 350 and 190 mµ as a function of pH, temperature, ionic strength, and solvent composition. Each form of carbonic anhydrase can exist in three different states: (a) native state, pH 6.5, 25°; (b) below pH 4, 25°; and (c) 8 m urea, 25°. The rotatory dispersion curves for these states differ considerably. In State a, all forms show a rotatory dispersion dominated by Cotton effects due to aromatic side chains. By comparing the protein optical rotatory dispersion to that of aromatic amino acids, contributions from both tryptophan and tyrosine side chains can be identified. Whereas the magnitude of tryptophan Cotton effects is constant for all forms of carbonic anhydrase, the tyrosine contributions vary considerably. In State b, all forms show a dispersion of simple polypeptide type with small helical contribution, human form B having the highest (approximately 12%) and bovine form B the lowest (4%). The rotation in both States a and b is different for each of the carbonic anhydrase forms investigated. Tentative conclusions regarding the helix content in native state favor a low helix content for all forms. In State c, all forms of the enzyme show a plain dispersion curve, with no trough above 220 mµ. The enhanced rotatory strength of aromatic transitions can, at present, best be explained as due to the perturbing effect from vicinal side chains arranged in an asymmetrical way around the aromatic chromophore.
Highlights
Cotton Effects in 250 to 350 mp Region and Ionic Strength Dependence of Rotation in V&sible Region-The first series of experiments was designed to examine in detail the structure of Cotton effects in the 250 to 300 rnp region for all forms of human and bovine carbonic anhydrase
The importance of this question lies in the fact that, if all the different forms of carbonic anhydrase, having different amino acid composition, were to show an effect of the same amplitude and shape, it could be ascribed to one specific detail of structure involving 1 or 2 residues common to all forms of carbonic anhydrase, possibly, but not necessarily, associated with the catalytic process
A minor contribution from the tyrosine residues cannot be excluded; on the contrary, we know that in principle all aromatic side chains contribute, the difference here being of magnitude and not of form
Summary
AND MATERIALSBovine carbonic anhydrase B was the electrophoretically homogeneous component described by Lindskog [5] and kindly supplied by Mr S. 0. Bovine carbonic anhydrase B was the electrophoretically homogeneous component described by Lindskog [5] and kindly supplied by Mr S. 0. Falkbring, Institut for Biokemi, University of Gothenburg, Sweden. Human carbonic anhydrase B and C, the electrophoretically homogeneous components as described by Nyman [6], were kindly supplied by him. The metal ion-free carbonic anhydrase human form C was prepared by dialysis of the crystalline enzyme as described by Tilander, Strandberg, and Fridborg [7]. The symbols used to designate different enzyme forms are those agreed upon by Lindskog, Nyman, and Edsall (see Reference 1)
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