The accessibility of protein-bound dinitrophenyl groups to univalent fragments of anti-dinitrophenyl antibody.
A series of N-(N-dinitrophenylaminoalkyl)maleimides were sythesized with alkyl-chain lengths of two, four and six carbon atoms. When these compounds reacted with the thiol group of mercaptalbumin, the tryptophan fluorescence of the protein was quenched. This change in fluorescence was used to determine the rate of reaction of the Dnp (dinitrophenyl)-maleimides with mercaptalbumin. The second-order rate constants were similar to those observed in reactions between low-molecular-weight thiol compounds and maleimides. When N-(N-Dnp-aminoalkyl)succinimidomercaptalbumins were added to univalent fragments of anti-Dnp antibody the antibody fluorescence was quenched. Florescence-quenching titrations showed that the protein-bound Dnp groups were fully available to the antibody even when the alkyl chain was short. The apparent dissociation constants were significantly greater than that of the interaction between anti-Dnp antibody and the free hapten, 6-(N-Dnp)-aminohexanoate. The antibody fluorescence was quenched efficienty by [dnp-Lys41]ribonuclease A, also with an increased dissociation constant. It could be concluded from the increase in dissociation constant that the Dnp group spent no more than 0.1% of its time in the dissociated state, available to antibody. The second-order rate constants for the association between the Dnp-mercaptablumins and the antibody were determined and were similar in magnitude to those observed in other interactions between protein and anti-protein antibody.
D000418:Albumins / Q000138:chemical synthesis; D000473:Alkanes / Q000138:chemical synthesis; D000614:Aminocaproates; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D004136:Dinitrobenzenes; D005455:Fluorescent Antibody Technique; D007128:Immunoglobulin Fragments / Q000302:isolation & purification; D007700:Kinetics; D008301:Maleimides / Q000138:chemical synthesis; D009578:Nitrobenzenes; D011485:Protein Binding; D012260:Ribonucleases; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis
D000418:Albumins; D000473:Alkanes; D000614:Aminocaproates; D002352:Carrier Proteins; D004136:Dinitrobenzenes; D007128:Immunoglobulin Fragments; D008301:Maleimides; D009578:Nitrobenzenes; D013438:Sulfhydryl Compounds; D012260:Ribonucleases