AFFINITY-CHROMATOGRAPHY OF YEAST ALCOHOL-DEHYDROGENASE USING IMMOBILIZED MONOCHLOROTRIAZINE COLORLESS COMPOUNDS

Four new symmetrical colourless biomimetics bearing two monochlorotria zine rings have been rationally designed and synthesized. These immobi lized colourless ligands and one analogue of Cibacron Blue F3GA on Sep harose CL-4B were used to purify alcohol dehydrogenase from baker's ye ast 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 sulphona tes as terminal rings, which is comparable to the result obtained from the analogue of Cibacron Blue F3GA. Differential spectra between liga nd-enzyme complexes and free ligands were studied. (C) 1997 by John Wi ley & Sons, Ltd.