Catalase hydroperoxide determination

L. Campanella, M.P. Sammartino, M. Tomassetti and S. Zannella, Hydroperoxide determination by a catalase OPEE application to the study of extra virgin olive oil rancidification process, Sens. Actuators B Chem., 76(1-3) (2001) 158-165. 

Concerning the mode of formation of ES, we prefer the concept that the substrate in a monolayer is chemisorbed to the active center of the enzyme protein, just as the experimental evidence pertaining to surface catalysis by inorganic catalysts indicates that in these reactions chemisorbed, not physically adsorbed, reactants are involved. Such a concept is supported by the demonstration of spectroscopically defined unstable intermediate compounds between enzyme and substrate in the decomposition by catalase of ethyl hydroperoxide,11 and in the interaction between peroxidase and hydrogen peroxide.18 Recently Chance18 determined by direct photoelectric measurements the dissociation con-... 

Hydroperoxides may be determined by measuring at 290 nm (e = 44100 M cm ) or 360 nm (e = 28000 cm ) the concentration of 13 formed in the presence of a large excess of ions. The reaction may be too slow for practical purposes, unless a catalyst is present. For example, an assay for lipid hydroperoxides conducted without a catalyst may require several measurements every 6 min until the absorbance reaches a maximum. Exclusion of air from the sample cuvette is important. The method is about 1000-fold more sensitive than thiosulfate titration The iodometric method with UVD at 360 was adopted for detecting the presence of hydroperoxides derived from protein, peptide or amino acid substrates subjected to y-radiation, after destroying the generated H2O2 with catalase. ... 

The kinetics of oxidation of Leuco Crystal Violet, depicted in equation 26 (Section III.B.2.a), can be applied for the determination of organic hydroperoxides after elimination of H2O2 with catalase . 

Figure 6.3 shows catalase transformation under the substrate (ROOH) effect in complex II to be the predominant pathway. For neutral substrates, which are hydroperoxides, the rate of complex II formation is independent of pH and is usually described by the second-order equation [103, 104], Complex II is the general intermediate for catalase and peroxidase reactions with the only difference that for catalase it is colored green (unpaired electron is localized on heme) and for peroxidase it is red (unpaired electron is localized on distal amino-acid fragment). Complex III is also colored red for peroxidase. However, the formation mechanism is different. Complexes II, III and IV are typical of peroxidases, whereas for catalase only complex II is formed. At the stage of complex II formation, the general properties and distinctive features of catalase and peroxidase were determined. 

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