Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody

Abstract

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.

Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury

“…The Dnp-pepstatins showed similar behaviour. In the presence of an excess of anti-Dnp antibody, which showed no change in Kapp on lowering the pH to 3.5 (Velick et al, 1960;Knight & Green, 1979), N-pepstatinyl-N'-Dnp-1,2-diaminoethane was bound less tightly by cathepsin D (Fig. 3).…”

“…0.2pM (with respect to binding sites) in 0.05 M-sodium phosphate buffer, pH 7.45, and the concentration of Dnp compound was 30-100,uM in Dnp groups. Corrections were made for dilution and for internal quenching during the titration (Knight & Green, 1979).…”

“…N6-Dnp-lysine (0), or N-pepstatinyl-N'-Dnp-1,6-diaminohexane (U), was added to anti-Dnp antibody (2ml, 0.22pM in Dnp-binding sites). Corrections were made for dilution and for internal quenching as described by Knight & Green (1979). The titration curve for N6-Dnp-lysine has been drawn.…”

Drugs as Haptens, Antigens, and Immunogens

“…Further work confirmed that in addition to the effects of the parent compounds, nitrophenylated cells and proteins were also potent immunogens [23,24] , although this was cell type and protein dependent. Antibodies generated in response to DNP-and TNPmodified proteins and peptides have been well studied and appear to be heterogenous in specificity and affinity for DNP [25][26][27] , although most react both to the modified amino acids and modified peptides.…”