Affinity chromatography of yeast alcohol dehydrogenase using immobilized monochlorotriazine colourless compounds

Four new symmetrical colourless biomimetics bearing two monochlorotriazine rings have been rationally designed and synthesized. These immobilized colourless ligands and one analogue of Cibacron Blue F3GA on Sepharose CL-4B were used to purify alcohol dehydrogenase from baker's yeast extract. Twenty-two-fold purification with 78% enzyme recovery was achieved in a single step with specific elution of NAD+ (5 mM) from the colourless absorbent comprised of two ortho-aminobenzene sulphonates as terminal rings, which is comparable to the result obtained from the analogue of Cibacron Blue F3GA. Differential spectra between ligand-enzyme complexes and free ligands were studied.