Interaction of bromophenol blue and related dyes with bovine neurophysin-I: use as a probe of neurophysin chemistry

Abstract

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.

NMR Study of Neurophysin Dimer Dissociation by Cosolvents

“…Interaction of the bromophenol blue with NP was first suggested by the fact that the degree of electrophoretic separation of different rat neurophysins was dependent on concentration of bromophenol blue as a tracking dye (Burtford and Pikering, 1972). One study showed that dye was bound predominantly to the dimeric form of the protein, based on ultracentrifugation studies (Carlson et al, 1981). However, another study suggested that dye thermodynamically prefers monomer, based on spectroscopically derived binding isotherms (Pearlmntter, 1979).…”

“…Titration of 2.7 mM NP-I with an increasing concentration of dye were carried out from 1:0.4to1:4 molar ratio at 25 oc and pH 2.3. At pH 2.3, NP-I has the highest affinity for bromophenol blue (Carlson et al, 1981). Figure 25 shows the spectrum of 1 mM bromophenol blue at pH 2.3.…”

“…The concentrations used in previous NP-dye studies were on the order of 10-2 mM (e.g. see Carlson et al, 1981 andPearlmntter, 1979). Nevertheless, our results may call for a re-evaluation of those studies and their validity.…”

“…Ultracentrifuge studies show that there is no species higher than dimer in solution at the concentrations used for NMR (Nicolas and Batelier, 1980), and the dimer ~ monomer equilibrium shifts to dimer as the pH decreases (Carlson and Breslow, 1981). As evidenced by CD results, no large secondary structure changes occur upon dimerization, but there are subtle changes in tertiary structure that involve the binding site for the ligand oxytocin or vasopression.…”

Molecular, Thermodynamic, and Biological Aspects of Recognition and Function in Neurophysin‐Hormone Systems: A Model System for the Analysis of Protein–Peptide Interactions

“…(46, SO)] and by marked similarities in most other properties. The single clear exception to this generality is the reaction of bovine neurophysin-I (oxytocin associated) with bromophenol blue, which is not paralleled by the vasopressin-associated bovine neurophysin-I1 (56,57). Rat neurophysins also appear to differ among themselves in reactivity towards bromophenol blue (58), but it has not been established whether this apparent difference between classes of neurophysins can be generalized.…”