The study of redox-active inorganic substituents of cellulase enzymes. Quarterly report, August 25--November 25, 1993

Attention is focused on the following: modification of CBHI with bis (2,2-bipyridine) ruthenium (II); and cellulase activity assay. CBHI was reacted with Ru(bpy) 2 (H{sub 2}O){sup 2+} at room temperature in 100 mM HEPES buffer (pH7.0) for 2 and 4 hours. The reaction vial was covered with aluminum foil to shield off light. Purified CBHI at a concentration of 1.5 mg/ml was generally employed, and a 10-fold excess ruthenium reagent was used. The reaction was quenched by diluting the reaction mixture with 50 mM acetate buffer (pH 5.0) and separating the excess Ru reagent using Amicon stirred filtration (yM-10) at 4 C. In parallel experiments the buffer was replaced with imidazole buffer 100 mM, pH 7.0, in order to block the axial ligand on the Ru(bpy)2 histidine. In a final exchange all buffers were replaced with acetate buffer in which the cellulase activity assay was carried out. UV/vis absorption spectra of native and modified CBHI were taken and compared