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A 350-and a 460-fold one step purification of alkaline phosphatase from a crude calf intestine extract has been achived using two new synthesized colourless biomimetic compounds coupled to Agarose CL-4B.

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. © 1997 John Wiley & Sons, Ltd.