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The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8·1012dm3mol-1s-1, and 1.6·1012dm3mol-1s-1, respectively.
The reactions of two heme peroxidases, horseradish peroxidase and lactoperoxidase and their compounds II (oxoferryl heme intermediates, Fe(IV)=0 or ferric protein radical Fe(III)R') and compounds III (resonance hybrids [Fe(IIIK)2 «-» Fe(II)-02l with superoxide radical anion generated enzymatically or radiolytically, and with hydroxyl radicals generated radiolytically, were investigated. It is suggested that only the protein radical form of compound II of lactoperoxidase reacts with superoxide, whereas compound II of horseradich peroxidase, which exists only in oxoferryl form, is unreactive towards superoxide. Compound III of the investigated peroxidases does not react with superoxide. The lactoperoxidase activity loss induced by hydroxyl radicals is closely related to the loss of the ability to form compound I (oxoferryl porphyrin n-cation radical, Fed V)=0(Por+) or oxoferryl protein radical Fe(IV)=0(R )). On the other hand, the modification of horseradish peroxidase induced by hydroxyl radicals has been reported to cause also restrictions in substrate binding (Gębicka, L. & Gębicki, J.L., 1996, Biochimie 78,62-65). Nevertheless, it has been found that only a small fraction of hydroxyl radicals generated homogeneously does inactivate the enzymes.
Cardiac ischemia/reperfusion leads to coronary endothelial dysfunction, mediated by superoxide anion (O2-), but not hydroxyl radical (.OH). Ischemic preconditioning and mitochondrial ATP-dependent potassium channel opener (diazoxide) protect endothelium in the mechanism involving attenuation of O2- burst at reperfusion. We hypothesize that the endothelial protection involves upregulation of myocardial anty-O2- defense. Langendorff-perfused guinea-pig hearts were subjected to global ischemia/reperfusion (IR) or were preconditioned prior to IR with three cycles of ischemia/reperfusion (IPC) or infusion/washout of 0.5 µM diazoxide. Coronary flow responses to acetylcholine were measures of endothelium-dependent vascular function. Myocardial outflow of O2- and of .OH during reperfusion and myocardial activities of superoxide dismutase (SOD) and catalase were measured. IR impaired acetylcholine response and augmented cardiac O2- and .OH outflow. IPC, diazoxide, and SOD (150 IU/ml) attenuated O2- outflow, increased .OH outflow and protected endothelium. There were no differences in Cu/Zn-SOD, Mn-SOD and catalase activities between sham-perfused and IR hearts and only catalase activity was increased in the IPC hearts. We speculate that: (i) IPC and diazoxide endothelial protection involves activation of some SOD-like anti-O2- mechanism resulting in attenuation of O2- burst and increase in .OH burst, (ii) improved SOD activity might have not been detected because it was confined to a small, although functionally important, enzyme fraction, like that bound to the endothelial glycocalyx.
 Although the term "antioxidant" is used very frequently, there are problems with the definition of antioxidants and estimation of antioxidant activity. The distinction between antioxidant and antiradical activities is not always obvious. This minireview discusses critically the principles, advantages and limitations of the most frequently used methods of estimation of antiradical and antioxidant activities.
Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (.OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating .OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce .OH. The addition of Fe2+ and Cu+ (0-20 µM) to KH resulted in a concentration-dependent increase in .OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 µM) did not result in .OH formation, these ions mediated significant .OH production in the presence of a number of reducing agents. The .OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 µM, 5 µM or 10 µM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in .OH formation. For each Fe2+ concentration tested, the .OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting .OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the .OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate .OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.
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