Discrimination between enantioselective and non-selective binding sites on cellobiohydrolase-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-blockers in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderma reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobilized on silica as stationary phases. The results revealed that the retention could be described by the function [equation; see text] where the observed capacity factor corresponds to the sum of an enantioselective 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 nondisplacable capacity factors (k'ns) for both enantiomers of the same drug. The experimental capacity factors (k'(x,C)) of the enantiomers all show a close fit to the adapted function. The Kd values calculated for the competitor were also virtually identical for each pair of enantiomers and were in accordance with Ki data determined for the competitors in classical enzyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar results were obtained with the resolution of rac-alprenolol and rac-metoprolol on CBH I phase.
Henriksson
H
H
Department of Biochemistry, Uppsala University, Biomedical Centre, Sweden.
Pettersson
G
G
Johansson
G
G
D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D020075:Phanerochaete / Q000201:enzymology; D013237:Stereoisomerism; D014242:Trichoderma / Q000201:enzymology
D008024:Ligands; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase