Bovine erythrocyte cupro-zinc protein. 2. Physicochemical properties and circular dichroism.

Abstract

The physicochemical properties and circular dichroism of bovine erythrocyte cupro‐zinc protein were investigated. In sedimentation velocity experiments in the ultacentrifuge, the protein sedimented as a single peak with an s°20,w of 3.06 S. The diffusion coefficient was 8.27 F. the partial specific volume 0.736 ml/g, and the instrinsic viscosity 0.033 (g/100 ml)‐1. Combination of these hydrodynamic data indicated a molecular weight near 33 000, which was in good agreement with chemical and gel filtration data. The molecular weight was also determined by the sedimentation equilibrium method using interference optics. By this method M̄w was 33 500, and the protein appeared to be homogeneous. The circular dichroism spectrum of bovine erythrocyte cupro‐zinc protein was unique among copper proteins in having positive contributions at 610, 440 and 350 nm, which disappeared on removing the metal atoms. In the ultraviolet there were positive contributions near 260, 270 and 290 nm, and removal of the metal atoms changed these into negative contributions at 265 and 285 nm and a positive contribution at 295 nm. Possible assignments of these bands are discussedd. In the far ultraviolet, the circular dichroism spectra of the native protein and apoprotein showed few differences, indicating that the metal atoms do not play a dominant role in determining protein conformation. However, urea had a profound effect on the circular dichroism of the apoprotein but little effect on that of the native protein. Neither the native protein nor the apoprotein showed the duble minimum between 200 and 220 nm characteristic of α‐helix, but rather the existence of a minimum at 210 nm suggested that the proteins contained some β‐pleated sheet structure. Reduction and carboxymethylation of thiol groups greatly reduced the ultraviolet circular dichroism, indicating that the disulphide bridges have a strong influence in determining the tertiary structure of the protein.