Interaction of bromophenol blue and related dyes with bovine neurophysin-I: use as a probe of neurophysin chemistry.
The interaction of bromophenol blue and related dyes with bovine neurophysin-I was studied by equilibrium dialysis and gel filtration, absorption and circular dichroism spectroscopy, and analytical ultracentrifugation. Binding isotherms for bromophenol blue showed positive cooperativity, with one strong site and one or more weaker sites present per polypeptide chain at pH 4 and an apparent increase in relative importance of the weaker sites of lower pH. Circular dichroism (CD) studies suggested displacement of bound dye by peptides that bind to the neurophysin hormone binding site. Titration of bound bromophenol blue indicated that the deprotonated dye was bound to the strong site with approximately 20-fold greater affinity than the protonated dye. The pH dependence of binding of bromophenol blue and of bromocresol purple, which has a higher pKa than bromophenol blue, indicated that binding was dependent on protonation of a protein residue with a pKa of 2.9. This residue was identified as a protein carboxyl, probably on an abnormal side chain, by studies of glycine ethyl ester modified neurophysin and carboxypeptidase-treated neurophysin. The presence of exciton interactions between bound dye molecules when only one dye was bound per polypeptide chain and analytical ultracentrifugation results indicated that dye was bound predominantly to the dimeric form of the protein. The implication of the data are discussed with respect to a kinetic model of dye-neurophysin interaction, used elsewhere in a study of neurophysin dimerization, that assumed interaction of protein monomers with protonated dye. Additionally, results are presented which suggest, in disagreement with conclusions based on the kinetic model, that there is a pH-dependent component of neurophysin dimerization which parallels low pH fluorescence and CD changes observed earlier.
D000818:Animals; D001665:Binding Sites; D001978:Bromphenol Blue; D002417:Cattle; D002942:Circular Dichroism; D004396:Coloring Agents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009481:Neurophysins; D010636:Phenols; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D004396:Coloring Agents; D009481:Neurophysins; D010636:Phenols; D001978:Bromphenol Blue