An analogue of the reactive textile dye, Cibacron Blue F3G-A, has been modified with nicotinamide to produce an artificial redox coenzyme, Blue N-3, whose properties reflect the dual presence of a blue anthraquinone chromophore and an N-substituted nicotinamide ring. The reduced coenzyme generated concomitantly with the oxidation of alcohols by horse liver alcohol dehydrogenase (HLADH) displayed difference absorbance (Delta lambda(max) 333 nm) and H-1-NMR spectra consistent with the presence of a 1,4-dihydro-nicotinamide ring. The artificial coenzyme mediated the HLADH-catalyzed oxidation of primary aliphatic and cyclic alcohols with pentan-1-ol being the best cosubstrate of those tested and exhibiting a turnover of 8.5% of that shown by NAD(+) at pH 9.0 and 25 degrees C. The apparent Michaelis constant (K-m for Blue N-3 (38 mu M) with ethanol as cosubstrate was similar to that of NAD(+) (10 mu M) determined with HLADH and the reaction followed a similar ordered mechanism. The relative catalytic efficiency [k(cat)/K-m(M(-1)s(-1))] of Blue N-3 was 0.4% of that shown by NAD(+) under these conditions. The artificial coenzyme also displayed activity with human liver beta(1) beta(1) alcohol dehydrogenase and sheep liver sorbitol dehydrogenase, two members of the same long-chain/polyol dehydrogenase family of enzymes. The artificial coenzyme appears to bind to these enzymes, inducing a conformational change and producing a catalytically competent complex in a manner similar to the natural coenzyme.