PROTEIN-BINDING CHIRAL DISCRIMINATION OF HPLC STATIONARY PHASES MADE WITH WHOLE, FRAGMENTED, AND 3RD DOMAIN TURKEY OVOMUCOID

Individual protein domains and two domains in combination were prepare d by enzymatic and chemical cleavage of turkey ovomucoid followed by i solation and purification by size-exclusion and ion-exchange chromatog raphy. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers amon g a wide range of racemates. The columns made from whole turkey ovomuc oid displayed chiral activity toward many racemates, where as a combin ation of the first and second domain resolved only a selected number o f aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exh ibited enantioselective protein binding for fused-ring aromatic weak a cids. Glycosylation of the third domain did not affect chiral recognit ion. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids r esponsible for binding. Molecular modeling of the ligand-protein compl exation provided insights into the ability of a protein surface to dis criminate enantiomers on the basis of multiple intermolecular interact ions.