Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody.
Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.
D001666:Binding Sites, Antibody; D055598:Chemical Phenomena; D002621:Chemistry; D004136:Dinitrobenzenes / Q000276:immunology*; D004139:Dinitrofluorobenzene / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007700:Kinetics; D009578:Nitrobenzenes / Q000276:immunology*; D010434:Pepsin A; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000276:immunology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D004136:Dinitrobenzenes; D007128:Immunoglobulin Fragments; D009578:Nitrobenzenes; D012710:Serum Albumin, Bovine; D004139:Dinitrofluorobenzene; D010434:Pepsin A