| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 89 === Microperoxidase-8 ( MP-8 ) is a water soluble model compound for the peroxidases. The kinetics for the reaction of MP-8 and sodium hypochlorite was studied by stopped-flow spectrophotometer.
At pH 10.7, the reaction of MP-8 with sodium hypochlorite results in a decrease of the absorbance at 397nm, the apperance of peak at 415, 520 and 550 nm in the difference spectrum. This indicates the transformation of MP-8 to the reaction intermediate compound II. At pH 7.0, the absorbance at 397nm rapidly decreases and returns to a lower value, but no absorbance change is observed at 415 nm in the difference spectrum. This suggests that compoundⅠwas formed and then converted back to native MP-8. As the production of the reaction intermediate reaches a maximum, the solution of intermediate in the age loop of stopped-flow spectrophotometer was mixed with the substrate, o-methoxyphenol. Reaction intermediates are quickly converted back to the native MP-8. The rate constant, k3, for the reaction of compound II and o-methoxyphenol is 1.37´106 M-1s-1 at pH 10.7. The production of compound II formed is favored at high pH. The catalytic pathway and the reaction intermediates formed are similar to those MP-8 reacting with hydrogen peroxide.
The rate constant for the formation of compound I at pH 10.7 is k1=1.1 ×105 M-1s-1, which is larger than that of obtained from the hydrogen peroxide-system. At pH 7.0, the rate constant is k1=2 ´106 M-1s-1. Besides, the rate constant for the formation of the organge product that from the substrate o-methoxyphenol at pH 7.0 gives the value of k1=6.8´106 M-1s-1, which is larger than that obtained from the hydrogen peroxide-system in the literature. The pH-dependence of rate of the product formation indicates that hypochlorous acid is the main oxidizing component.
The pH-dependence of chemiluminescence intensity for the luminol-hypochlorous acid system is bell-shaped with the maximum intensity is occured at pH 10.5. β-cyclodextrin enhances the chemiluminescence intensity and chemiexcitation efficiency at physiological pH. 9 mM β-CD enhances the chemiluminescence intensity by a factor up to 30 at pH 7.5. No increase in intensity at alkaline pH was observed. The inclusion of the luminol molecule or the reaction intermediate in the interior cavity of the β-cyclodextrin is proposed for the observed enhancement.
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