Investigation Of A 6-Fluorotryptophan Substituted Scfv

For many years our laboratory has pursued an understanding of the protein characteristics which confer specificity and affinity to the antibody for its antigen using a family of monoclonal antibodies to hen egg white lysozyme (HyHEL26, 10, 8 and 63, primarily.) We find that the binding is best characterized by a two-step model representing an association complex becoming a docked complex, evidencing a conformational change. In a recently produced scFv variant of HyHEL10 in which all the tryptophans were substituted with the 6-fluoro form we studied kinetic behavior by Biacore SPR, using our usual protocol to obtain kinetic characterization. We observed that the affinity to lysozyme was concentration dependant, though it did not reflect oligomerization; it changes gradually, allowing investigation, decreasing by an order of magnitude over a period of 3 hours and that most of the change is due to the decrease in the docking step. This repeatable behavior is reversed upon sample reconcentration and delayed by cold. To explore the possible role of folding or water movement we investigated the impact of TMAO, glycerol and some detergents. We also did further exploration by SPR, fluorescence spectroscopy, and other biophysical characterizations in order to better understand the molecular events responsible for this dramatic affinity change.