Application of Molecular Absorption Properties of Horseradish Peroxidase for Self-Indicating Enzymatic Interactions and Analytical Methods

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• 作者：Vanesa Sanz ; Susana de Marcos ; Juan R. Castillo ; and Javier Galbá ; n
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：January 26, 2005
• 年：2005
• 卷：127
• 期：3
• 页码：1038 - 1048
• 全文大小：340K
• 年卷期：v.127,no.3(January 26, 2005)
• ISSN：1520-5126

文摘

In this paper an in depth study is presented of the use of the horseradish peroxidase (HRP)enzyme as a self-indicating biorecognition reagent in UV-vis molecular absorption spectrometry. The HRP/H₂O₂ reaction mechanism in the absence of an external substrate has been clarified, and the interactionbetween HRP and glucose oxidase (GOx) has been studied. It has been demonstrated that GOx can actas a substrate of HRP; in both cases the kinetic constants have been obtained and mathematical modelshave been developed. Second, the HRP/H₂O₂ reaction is used to follow a H₂O₂-producing enzymaticreaction, the glucose reaction with GOx being used as a model. As an application of this, two methodologieshave been proposed for glucose determination: with or without previous incubation of glucose with GOx.In both cases mathematical models relating HRP absorbance changes to glucose concentration have beendeveloped and tested; both methods have been optimized, analytically characterized, and tested for glucosedetermination in samples. The methodology described could be applied to other heme-proteins and toother H₂O₂-producing enzymatic reactions. The models permit the reaction constants to be calculated.From the analytical chemistry point of view the models allow the prediction of the method sensitivity forother analytes involved in this type of reaction if the kinetic constants are known and can be used in thedesign of optical sensors.