Denaturation and leaching study of horseradish peroxidase encapsulated in sol-gel matrices

Sol-gel matrices have been shown to be relatively inert while preserving the spectroscopic properties and biological activity of the encapsulated proteins. Horseradish peroxidase (HRP) is a hemeprotein widely used in the field of biosensors because of its high specificity for hydrogen peroxide. However, partial inactivation of the protein has been reported when incorporated in aged gels. Whether that inactivation comes from the unfolding of some of the encapsulated proteins or from the leaching of the heme non-covalent active site of HRP is evaluated by absorption and fluorescence spectroscopy. This study shows that the single Trytophan (Trp) fluorescence of HRP may be used to distinguish denaturation processes from leaching of the heme group, as well as to estimate the extent of the denaturation.