Dye Affinity Labelling of Yeast Alcohol Dehydrogenase

Abstract

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°C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (k_obs) exhibited a non-linear dependence on VBAR concentration from 22 to 106nmol, with a maximum rate of inactivation (k₃) of 0.134min^?1 and k_D of 141.7 μM. 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.