RESUMEN
Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination, which was followed by passing the digest through a 1 kDa equipped tangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate the CPH into four peptide fractions (CF1-CF4) and their in vitro antioxidant properties investigated. Results showed that most of the peptide fractions (CF2-CF4) displayed significantly higher (p<0.05) oxygen radical absorbance capacity values (698-942 µM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17-32%) than those of CPH (613 µM TE/g and 19%, respectively). However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxyl radicals as well as significantly higher (p<0.05) ferric iron reduction and chelation of iron than the RP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acid oxidation.
Asunto(s)
Antioxidantes/farmacología , Proteínas de Peces/farmacología , Gadus morhua/metabolismo , Péptidos/farmacología , Hidrolisados de Proteína/farmacología , Aminoácidos/análisis , Animales , Quelantes/farmacología , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Proteínas de Peces/análisis , Depuradores de Radicales Libres/farmacología , Técnicas In Vitro , Ácido Linoleico/química , Hidrolisados de Proteína/análisisRESUMEN
This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram-negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram-negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane. By using immunoelectron microscopy of Ptm-treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis.