Dye affinity labelling of yeast alcohol dehydrogenase

The interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC. Incubation of ADH with purified VBAR at pH 8.0 and 37 degrees C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inactivation (k3) of 0.134 min-1 and kD of 141.7 microM. The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+. These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH.