Structural Properties of Stellacyanin, a Copper Mucoprotein from Rhus vernicifera, the Japanese Lac Tree

Abstract

Stellacyanin, or Rhus vernicifera blue protein, was purified from lac acetone powders. It is 20% carbohydrate and 20% hexosamine with the remainder being a polypeptide matrix of 108 amino acid residues in which is embedded a single cupric atom. The molecule contains no methionine or valine. Optical rotatory dispersion and optical and electron paramagnetic resonance (EPR) spectral studies show that the ligand field of the copper of stellacyanin contains large odd and rhombic components and is thereby distorted away from the usual square-planar axial configuration. This results in an unusual EPR spectrum and an extraordinarily intense blue color. Raising the pH of a stellacyanin solution above 8.3 relaxes the rhombic distortions. This leads to downward shifts in wave lengths of the peaks in the visible and near ultraviolet optical spectrum, and the EPR spectrum changes to that of basic copper bis-biuret. The pH may be returned from 11.5 to 8.3, restoring the original optical and EPR spectra. A pH above 11.5 produces irreversible structural changes within the molecule. It is concluded from this stepwise denaturation that the close ligands of copper in stellacyanin are 4 nitrogen atoms. Lowering the pH of a stellacyanin solution below 2.0 releases the odd constraints and the optical absorption decreases. With longer incubation the copper becomes more accessible to ethylenediaminetetraacetate so that raising the pH to neutrality does not effect a reconstitution of color. Ultraviolet optical rotatory dispersion studies indicate that stellacyanin contains little or no helical structure.