RESUMEN
In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe...S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A(695)) were compared. A strict concurrency between Fe...S(Met80) breaking (A(695)) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A(695) completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A(695) did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe...S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe...S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H(2)O(2) as a result of the hydrophobic interaction between cardiolipin and cytochrome.
Asunto(s)
Cardiolipinas/farmacología , Citocromo-c Peroxidasa/metabolismo , Animales , Bovinos , Activación Enzimática , Peróxido de Hidrógeno/metabolismo , Hierro/química , Liposomas/metabolismo , Mediciones Luminiscentes , Metionina/química , Concentración Osmolar , Dodecil Sulfato de Sodio/farmacología , EspectrofotometríaRESUMEN
In this work, the effect of liposomes consisting of tetraoleyl cardiolipin and dioleyl phosphatidylcholine (1 : 1, mol/mol) on the rate of three more reactions of Cyt c heme with H2O2 was studied: (i) Cyt c (Fe2+) oxidation to Cyt c (Fe3+), (ii) Fe...S(Met80) bond breaking, and (iii) heme porphyrin ring decomposition. It was revealed that the rates of all those reactions increased greatly in the presence of liposomes containing cardiolipin and not of those consisting of only phosphatidylcholine, and approximately to the same extent as peroxidase activity. These data suggest that cardiolipin activates specifically Cyt c peroxidase activity not only because it promotes Fe...S(Met80) bond breaking but also facilitates H2O2 penetration to the reaction center.
Asunto(s)
Cardiolipinas/farmacología , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Peróxido de Hidrógeno/metabolismo , Animales , Sitios de Unión , Activación Enzimática , Compuestos Ferrosos/metabolismo , Hierro/química , Liposomas , Metionina/química , Oxidación-ReducciónRESUMEN
Based on the optimized spectrophotometric determination of pyrogens (of various classes ( p-aminophenol and endotoxins), thermal lensing was applied to the determination of these substances at the submicrogram level. The limit of detection of p-aminophenol, a pyrogenic impurity in pharmaceutical formulations of paracetamol, by reaction with resorcinol in alkaline solutions is 100 ng mL(-1). Phloroglucinol was considered as an analog of resorcinol as a reagent in this reaction. The conditions of spectrophotometric determination of pyrogenic lipopolysaccharides (endotoxins) by ion-pair formation with methylene blue (the limit of detection is 100 ng mL(-1)), by ion-pair formation with Stains-All (1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide) (the limit of detection is 500 ng mL(-1)), and by reaction of 2-keto-3-deoxyoctonic acid with thiobarbituric acid (the limit of detection is 800 ng mL(-1)) were proposed. The optimized procedure for 2-keto-3-deoxyoctonic acid was applied for thermal lensing that provided a decrease in the limit of detection to 70 ng mL(-1) and was also used for lipopolysaccharide determination in the endotoxin standard from E. coli.