Dye affinity labelling of yeast alcohol dehydrogenase

The interaction df yeast alcohol dehydrogenase (ADH) with the reactive chlo rotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to h omogeneity 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 (k(obs)) exhibited a non-linear depend ence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inac tivation (k(3)) Of 0.134 min(-1) and k(D) of 141.7 mu M. The inhibition was irreversible and activity could not be recovered by gel-filtration chromat ography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD(+). These results suggest that VBAR acts as an af finity label at the nucleotide binding site of yeast ADH.