Studies on the chemical modification of the tyrosine residue in bovine neurophysin-II.
1. Bovine neurophysin-II contains 1mol of tyrosine residue/10000g of protein. This residue could be readily nitrated with tetranitromethane. On hydrolysis and amino acid analysis 1mol of 3-nitrotyrosine was found/10000g of protein. Starchgel electrophoresis at pH8.5 showed that nitration had converted the native protein into a single, more acidic species. The increase in acidity was consistent with the observed fall in pK of the tyrosine hydroxyl from 9.2 in native neurophysin to 7.3 in the nitrated protein. Further, the absence of any intermediate species, even under conditions of minimum substitution, confirmed that the molecular weight of the monomer is 10000. 2. O-Acetylation of the tyrosine residue was carried out with N-acetylimidazole, in conjunction with the reversible blocking of amino groups by citraconylation. The degree of O-acetylation, determined spectroscopically, was 0.9mol of O-acetyltyrosine/10000g of protein. 3. The hormone-binding ability of modified protein was tested by equilibrium dialysis and was found to be unchanged by either nitration or O-acetylation of the tyrosine residue. 4. Interaction of neurophysin-II and [8-arginine]-vasopressin gave rise to a characteristic difference spectrum with a peak at 286.8nm and shoulder at 279.6nm. Part of this hyperchromicity is thought to result from entry of the tyrosine residue at position 2 in the hormone into the hydrophobic environment of the binding site. With nitrated neurophysin-II a second peak appeared at 436nm, showing that the tyrosine of the protein is also perturbed. The very large red shift (84nm) in this region suggests that the 3-nitrotyrosyl residue not only enters a more hydrophobic environment on protein-hormone interaction, but is caused to ionize more fully by the approach of some positively charged group.
D000473:Alkanes; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003956:Dialysis; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010909:Pituitary Hormones, Posterior / Q000032:analysis* / Q000494:pharmacology; D013053:Spectrophotometry; D014443:Tyrosine / Q000032:analysis*; D014667:Vasopressins / Q000494:pharmacology
D000473:Alkanes; D000596:Amino Acids; D010909:Pituitary Hormones, Posterior; D014667:Vasopressins; D014443:Tyrosine