Application of Electron Paramagnetic Resonance Spectroscopy for Validation of the Novel (AN+DN) Solvent Polarity Scale

Based on solvation studies of polymers, the sum (1:1) of the electron acceptor (AN) and electron donor (DN) values of solvents has been proposed as an alternative polarity scale. To test this, the electron paramagnetic resonance isotropic hyperfine splitting constant, a parameter known to be dependent on the polarity/proticity of the medium, was correlated with the (AN+DN) term using three paramagnetic probes. The linear regression coefficient calculated for 15 different solvents was approximately 0.9, quite similar to those of other well-known polarity parameters, attesting to the validity of the (AN+DN) term as a novel “two-parameter” solvent polarity scale

Effect of polysaccharide capsule of the microalgae Staurastrum iversenii var. americanum on diffusion of charged and uncharged molecules, using EPR technique

The existence of a mucilaginous envelope, sheath or capsule is usual in many desmids, but few data concerning its function are available. Previous studies of the transport function and permeation of molecules through the algae capsules were done using the algae Spondylosium panduriforme and Nephrocytium lunatum, the Electron Paramagnetic Resonance (EPR) technique, and different spin labels. The results suggested that the capsule functions as a selective diffusion medium. In the present work charged and uncharged molecules (spin labels group A) and Staurastrum iversenii var. americanum (Desmids),whose alga presents a great mucilaginous capsule, were used. Charged nitroxide molecules similar to amino acids (spin labels group B) were also used allowing a better understanding of the electrostatic effect in the permeation process across the capsule. The role of the cell capsule in the solute diffusion was evaluated by determining the capsulated and decapsulated cell permeation times. The permeation times for all spin labels tested in the cells lacking capsules were always shorter than those containing this physical barrier. The decay times of spin labels group A observed for S. iversenii were compared to other studied algae. The results regarding the diffusion of charged spin labels group B suggested that the interaction of cell capsule occurs more strongly with negatively charged molecules than with positively charged ones. The results obtained in this work with spin labels group A confirm that the capsule is an essential structure for the cell, and that due to the polar interactions with the spin labels, it plays an important role in the selection of small molecules. Several parameters, mainly those of electrostatic nature, seem to control the permeation across the algal capsules of spin labels group B, showing that structures which are similar to amino acids could diffuse across the interior of the algal cell.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)USP Instituto de Física de São Carlos Departamento de Física e InformáticaUniversidade Federal de São Carlos Departamento de BotânicaUniversidade Federal de São Paulo (UNIFESP) Departamento de BiofísicaUNIFESP, Depto. de BiofísicaSciEL

The angiotensin II AT(1), receptor structure-activity correlations in the light of rhodopsin structure

The most prevalent physiological effects of ANG II, the main product of the renin-angiotensin system, are mediated by the AT(1) receptor, a rhodopsin-like AGPCR. Numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation, which were complemented by site-directed mutagenesis studies on the AT(1) receptor to investigate the role of its structure in ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. the knowledge of the high-resolution structure of rhodopsin allowed homology modeling of the AT(1) receptor. the models thus built and mutagenesis data indicate that physiological (agonist binding) or constitutive (mutated receptor) activation may involve different degrees of expansion of the receptor's central cavity. Residues in ANG II structure seem to control these conformational changes and to dictate the type of cytosolic event elicited during the activation. 1) Agonist aromatic residues (Phe(8) and Tyr(4)) favor the coupling to G protein, and 2) absence of these residues can favor a mechanism leading directly to receptor internalization via phosphorylation by specific kinases of the receptor's COOH-terminal Ser and Thr residues, arrestin binding, and clathrin-dependent coated-pit vesicles. On the other hand, the NH2-terminal residues of the agonists ANG II and [Sar(1)]-ANG II were found to bind by two distinct modes to the AT(1) receptor extracellular site flanked by the COOH-terminal segments of the EC-3 loop and the NH2-terminal domain. Since the [Sar(1)]-ligand is the most potent molecule to trigger tachyphylaxis in AT(1) receptors, it was suggested that its corresponding binding mode might be associated with this special condition of receptors.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilUniv São Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, BR-14049 Ribeirao Preto, BrazilUniv São Paulo, Inst Chem, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilWeb of Scienc

Alternative and simple normal-phase HPLC enantioseparation of a chiral amino acid-type spin label derivative

In this work an alternative chromatographic process was developed for fractionating the (+)-(3R,4R) and ( - )-(3S,4S) enantiomers of the chiral trans β-amino acid trans-2,2,5,5-tetramethylpyrrolidine-3-amino-4-carboxylic acid (POAC), which was protected at its amine group for further coupling to a peptide, polymer or other macromolecule. The HPLC enantioseparation was achieved using a chiral cellulose-based normal stationary phase and isocratic elution. The n-hexane:isopropanol system, always with greater amount of the former component, was used as mobile phase as revealed by improved fractionation property of both components, demonstrated by the separation factor and resolution index values. These parameters presented values of 3.7 and 18.4 and of 2.0 and 6.7 when in 90:10 (v/v) and 80:20 (v/v) n-hexane:isopropanol solutions, respectively. These findings indicate that the one-step chromatographic purification strategy using normal-phase is feasible, thus opening the perspective of a fast large-scale production this paramagnetic spin probe.Neste trabalho desenvolveu-se um processo alternativo para separação cromatográfica dos enantiômeros (+)-(3R,4R) e ( - )-(3S,4S) do β-aminoácido quiral trans-2,2,5,5-tetrametilpirrolidina-3-amino-4-carboxílico (POAC), que estava protegido no grupo amínico para posterior ligação a um peptídeo, polímero ou outra macromolécula. A enantioseparação foi obtida por HPLC usando uma fase estacionária normal à base de celulose quiral e eluição isocrática. O sistema n-hexano:isopropanol, sempre com maior quantidade do primeiro solvente, foi usado como fase móvel, pois forneceu os melhores resultados na separação dos dois componentes, constatado pelos valores mais elevados de fator de separação e de índice de resolução cromatográfica. Estes parâmetros apresentaram valores de 3,7 e 18,4 e de 2,0 e 6,7 nas soluções com proporção 90:10 (v/v) e 80:20 (v/v) de n-hexano:isopropanol, respectivamente. Estes dados indicam que a estratégia de purificação cromatográfica em uma única etapa usando fase normal é viável, abrindo assim a perspectiva de uma produção rápida e em grande escala desta sonda paramagnética.18401845Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. the ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac(0)-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac, Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for a-MSH, (C) 1999 Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-01498 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-01498 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides

Similar to melanocyte stimulating hormone (alpha -MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha -MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides, Correlation times, calculated from tire electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-66318 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-11176 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc