Discrimination between enantioselective and non-selective binding sites oncellobiohydrolase-based stationary phases by site specific competing ligands

A systematic study was performed to investigate the influence of cellobiose or lactose on the enantioselective retention behaviour of some beta-blocke rs in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderm a reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobiliz ed on silica as stationary phases. The results revealed that the retention could be described by the function k(x)' = k(ns,x)' x k(es,x)'/1 + [competitor]/K-d where the observed capacity factor corresponds to the sum of an enantiosele ctive mode being influenced by a site specific competing ligand (competitor ) and a non-selective mode unaffected by the competitor. A non-constrained non-linear least-square regression gave in all cases virtually identical no ndisplacable capacity factors (k(ns)') for both enantiomers of the same dru g. The experimental capacity factors (k(x, C)') of the enantiomers all show a close fit to the adapted function. The K-d values calculated for the com petitor were also virtually identical for each pair of enantiomers and were in accordance with K-i data determined for the competitors in classical en zyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar r esults were obtained with the resolution of rac-alprenolol and rac-metoprol ol on CBH I phase. (C) 1999 Elsevier Science BN. All rights reserved.