Effects of secondary forces on the primary antibody-ligand interaction.
The binding efficiency of high affinity monoclonal antifluorescyl antibody 4.4-20 with the homologous ligand situated in different protein environments has been investigated to quantitate the effect of non-active site secondary factors. To synthesize monofluoresceinated proteins, fluorescein 5-isothiocyanate was reacted with a 100-fold molar excess of ribonuclease, lysozyme, lactalbumin and bovine serum albumin. Absorption and emission spectra, as well as fluorescence life-time measurements which yielded discrete components and proteolytic studies suggested that fluorescein was conjugated to a specific lysine residue consistent with a non-random distribution of lysines within each protein population. The derivatized residue was probably a surface moiety based on accessibility analyses with iodide as a dynamic quencher. Dissociation rate analyses indicated that the relative release time of 4.4-20 with each monofluoresceinated protein was Fl-RNAse > or = Fl-lyso > or = FDS > Fl-lact > or = Fl-BSA which correlated with changes in free energy of binding. Relative fluorescence quenching measurements of the fluorescein moiety indicated that 4.4-20 showed decreasing quenching in the order FDS > Fl-RNAse > Fl-lyso > or = Fl-lact > Fl-BSA. Because spectral data indicated that fluorescein was conjugated to a specific residue or a non-random distribution of residues in each protein population, the results represented the effect of a single distinct environment or a weighted average of different microenvironments. Results have been interpreted within the theoretical framework of a dynamic antibody model involving conformer selection and the relative effects of primary and secondary interactions.
Mummert
M E
ME
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Voss
E W
EW
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D002417:Cattle; D003198:Computer Simulation; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D006801:Humans; D008024:Ligands
D000906:Antibodies; D005452:Fluoresceins; D008024:Ligands; D019793:Fluorescein