New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as term inal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-m ethyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbe nts, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, wer e studied for their purifying ability for the glutathione-recognising enzym es, NAD(+)-dependent formaldehyde dehydrogenase (FaDH) from Candida boidini i, NAD(P)(+)-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I(GSTI). All biomimetic adsorbe nts showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent c omprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exh ibited the highest purifying ability for FaDH and GSTI, whereas, the affini ty adsorbent comprising as terminal biomimetic moiety methyl-glutathione (B M2) exhibited the highest purifying ability for GSHR. The BMI adsorbent was integrated in a facile two-step purification procedure for FaDH. The purif ied enzyme showed a specific activity equal to 79 U/mg and a single band af ter sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Mole cular modelling was employed to visualise the binding of BMI with FaDH, ind icating favourable positioning of the key structural features of the biomim etic dye. The anthraquinone moiety provides the driving force for the corre ct positioning of the glutathionyl-biomimetic moiety in the binding site. I t is located deep in the active site cleft forming many favourable hydropho bic contacts with hydrophobic residues of the enzyme. The positioning of th e glutathione-like biomimetic moiety is primarily achieved by the strong io nic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydropho bic contacts made with Tyr 92 and Met 140. Molecular models were also produ ced for the binding of BM1 and BM3 (glutathione-substituted) to GST I. In b oth cases the biomimetic dye forms multiple hydrophobic interactions with t he enzyme through binding to a surface pocket. While the glutathioine moiet y of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better pu rification results achieved with BM1. (C) 2001 Elsevier Science B.V. All ri ghts reserved.