Acid–Base Titration of Melanocortin Peptides: Evidence of Trp Rotational Conformers Interconversion Roberto M. Fernandez, 1 Renata F. F. Vieira, 2 Clo ´ vis R. Nakaie, 2 M. Teresa Lamy, 1 Amando S. Ito 3 1 Instituto de Fı´sica, Universidade de Sa ˜ o Paulo, Sa ˜ o Paulo, SP, Brazil 2 Departamento de Biofı´sica, Universidade Federal de Sa ˜o Paulo, Sa ˜ o Paulo, SP, Brazil 3 Faculdade de Filosofia Cie ˆ ncias e Letras de Ribeira ˜o Preto, Universidade de Sa ˜ o Paulo, Ribeira ˜ o Preto, SP, Brazil Received 28 January 2004; revised 13 July 2004; accepted 26 October 2004 Published online 18 January 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20210 Abstract: Tryptophan time-resolved fluorescence was used to monitor acid– base titration prop- erties of -melanocyte stimulating hormone (-MSH) and the biologically more potent analog [Nle 4 , D-Phe 7 ] -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluores- cence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+  1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans  1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in -MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac- labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain Correspondence to: Amando S. Ito; e-mail: amandosi@ ffclrp.usp.br Contract grant sponsor: FAPESP and CNPq Biopolymers (Peptide Science), Vol. 80, 643– 650 (2005) © 2005 Wiley Periodicals, Inc. 643