Reactivation of peroxidase activity in human saliva samples by polyphenols.

OBJECTIVES: The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN-) to hypo-thiocyanite (-OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. DESIGN: Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H2O2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived -OSCN production. RESULTS: Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The -OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H2O2-induced inactivation. CONCLUSION: The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Tannins and Tannin-Related Derivatives Enhance the (Pseudo-)Halogenating Activity of Lactoperoxidase.

Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo-)halogenating activity, i.e., the oxidation of SCN- to hypothiocyanite -OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2-nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated -OSCN formation. The results were combined with docking studies and molecular orbital analysis. The -OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo-)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, the results provide new insights into the antibacterial mode of action of tannins and related compounds. Furthermore, chemical properties of the tested compounds that are important for efficient enzyme-substrate interaction and regeneration of the -OSCN formation by LPO were identified.

Flavonoids as promoters of the (pseudo-)halogenating activity of lactoperoxidase and myeloperoxidase.

In this study several flavonoids were tested for their potential to regenerate the (pseudo-)halogenating activity (hypothiocyanite formation) of the heme peroxidases lactoperoxidase (LPO) and myeloperoxidase (MPO) after hydrogen peroxide-mediated enzyme inactivation. Several flavonoid subclasses with varying hydroxylation patterns (especially of the flavonoid B-ring) were examined in order to identify structural properties of efficient enzyme regenerators. Kinetic parameters and second-order rate constants were determined. A 3',4'-dihydroxylated B-ring together with C-ring saturation and hydroxylation were found to be important structural elements, which strongly influence the flavonoid binding and oxidizability by the LPO/MPO redox intermediates Compounds I and II. In combination with docking studies these results allow an understanding of the differences between flavonoids that promote the hypothiocyanite production by LPO and MPO and those that inhibit this enzymatic reaction.

New insights into thiocyanate oxidation by human myeloperoxidase.

Human myeloperoxidase (MPO) uses chloride and thiocyanate as physiological substrates at neutral pH. Oxidation of thiocyanate to hypothiocyanite mediated by the redox intermediate Compound I rapidly restores the ferric state of MPO. At low thiocyanate concentration and in the presence of hydrogen peroxide the observed reaction sequence is Compound I→ferric MPO→Compound II→MPO-cyanide complex, whereas at high thiocyanate concentrations and in the absence of H2O2 the only observed transition is Compound I→ferric MPO. The reaction of ferric MPO with hypothiocyanite directly forms the MPO-cyanide complex, whereas a transient product derived from the reaction between hypothiocyanite and hydrogen peroxide is demonstrated to mediate the conversion of ferric MPO to Compound II. Mechanisms for those reactions are discussed and proposed.

Lactoperoxidase as a potential drug target.

INTRODUCTION: Lactoperoxidase (LPO) belongs to the immunologically relevant mammalian heme peroxidases. The enzyme contributes in external secretions to the humoral immune defense against pathogens by oxidation of thiocyanate (SCN(-)) and iodide (I(-)). The generation of oxidized thiocyanate and/or iodine species is also important in numerous biotechnological applications of LPO. AREAS COVERED: In this review, we give an overview about the present knowledge of LPO concerning enzymatic structure, catalytic cycles and (pseudo-)halogenated species generated by the enzyme. Redox properties of LPO as well as kinetic aspects regarding the different enzymatic cycles are discussed in order to gain insights into the disturbance of the (pseudo-)halogenating enzyme activity under pathological conditions. Important structural features of LPO and crystallographic studies on the interaction and reaction of organic substrates with the enzyme are also summarized. A broad discussion is devoted to the binding and oxidation of substrates that either inhibit or promote LPO activity. EXPERT OPINION: On the basis of these data, different strategies to further optimize LPO functions in humoral defense of mucous surfaces and biotechnological applications are discussed. In particular, hydrophobic organic substrates with a 3,4-dihydroxyphenyl partial structure considerably enhance the (pseudo-)halogenating activity of LPO. Their application provides, thus, a new strategy to enhance the anti-microbial activity of this enzyme.

Enhancing hypothiocyanite production by lactoperoxidase - mechanism and chemical properties of promotors.

BACKGROUND: The heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. METHODS: The results were obtained by measuring enzyme-kinetic parameters using UV-vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. RESULTS: In this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such as hydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. CONCLUSIONS: This study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. SIGNIFICANCE: This study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme.