The binding sites of proteins such as antibodies are known to often contain tryptophan (Trp) residues, whose fluorescent properties may be altered upon ligand binding. Conformational changes within the binding site or simply the presence of the ligand can result in either fluorescence quenching or enhancement. Highly stereoselective antibodies to amino acids have been used in a variety of analytical techniques for the sensitive detection of enantiomeric impurities and for enantiomer separation.
Here, we show how tryptophan fluorescence was used to determine the affinity of an anti-D-amino acid antibody to a variety of standard and non-standard amino acids.
The BMG LABTECH multi-mode plate reader detected tryptophan fluorescence and its dependence on pH and temperature with high sensitivity. Furthermore, Trp-fluorescence reported on stereoselectivity of a phenylalanine antibody that selectively bound D-phenylalanine (increase in fluorescence) while no binding (constant fluorescence) was observed for L-phenylalanine.