Fluorescence studies of native and modified neurophysins. Effects of peptides and pH

• Published in: Biochemistry (Easton) Vol. 18; no. 6; pp. 1026 - 1036
• Main Authors: Sur, Shyamali Sengupta, Rabbani, Lillian D, Libman, Lawrence, Breslow, Esther
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.03.1979
• Subjects: Animals; Cattle; Hydrogen-Ion Concentration; Neurophysins; Oxytocin; Protein Binding; Spectrometry, Fluorescence; Vasopressins
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00573a015

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by Förster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.