RESUMEN
A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d1/acetic acid-d4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent). Mixtures of D- and L-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of D-/L-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the alpha-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Péptidos/aislamiento & purificación , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Aminoácidos/química , Deuterio , Dinitrobencenos/química , Hidrólisis , Indicadores y Reactivos , Datos de Secuencia Molecular , Péptidos/química , Conformación ProteicaRESUMEN
Equine platelets, when treated with the anthelmintic drug diethylcarbamazine (DEC), gave a dose-dependent release of radiolabeled serotonin without concomitant aggregation. At levels of the drug that gave only minimal release of radiolabel, marked dose-dependent inhibition of platelet aggregation to three of four platelet agonists tested--adenosine diphosphate (ADP), collagen, and arachidonic acid--was observed. With ADP, inhibition was observed to be reversed by removal of DEC prior to agonist challenge. However, with collagen, inhibition was only partially reduced by prior removal of DEC; whereas with arachidonate the DEC inhibition appeared not to be reduced by removal of the drug. Thrombin-induced aggregation was not inhibited by DEC. DEC therefore has the heretofore unrecognized property of modulating platelet function to several platelet agonists as well as inducing the platelet release of serotonin. Our results would suggest a reversible membrane-drug interaction as the potential site of modulation for ADP and collagen, whereas an apparent irreversible inhibition is suggested for arachidonate-induced aggregation.