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

Abstract

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.

Keywords: 3,4-dihydroxylated compounds; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); Aromatic compounds; DB, double bond; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); EPO, eosinophil peroxidase; Hypothiocyanite; Inflammation; LPO, lactoperoxidase; Lactoperoxidase; MPO, myeloperoxidase; Peroxidases; SB, single bond; TNB, 5-thio-2-nitrobenzoic acid.; ssp., subspecies.

Fig. 1

Lactoperoxidase-mediated TNB degradation. The effect of low and high H₂O₂ concentrations on the (pseudo-)halogenating enzyme activity was tested at 37 °C and pH 7.4. Thereby (pseudo-) physiological conditions in the oral cavity were imitated by using 5 nM LPO and 2 mM SCN⁻. The columns correspond to the sole presence of H₂O₂ (white), the additionally presence of LPO (gray) and the whole LPO–H₂O₂–SCN⁻ system (black), respectively. The fourth column (light gray) shows the corresponding net effect of the ⁻OSCN mediated TNB degradation. At higher H₂O₂ concentrations a significant lower net LPO activity was found. Mean and standard deviation of n=3–6 experiments are given.

Fig. 2

Example for the modulation of the LPO-mediated TNB degradation. The effect of eriodictyol was tested at 37 °C and pH 7.4 using 5 nM LPO, and 2 mM SCN⁻ (continuous lines). The reaction was started by adding 80 µM H₂O₂ (arrow). As shown in (A) while in the absence of the flavonoid (light gray) only a slow drop in the absorbance was observed, 0.1 µM (gray) and 5 µM (black) eriodictyol considerably accelerated the TNB degradation. This effect was not observed in the absence of SCN⁻ (dashed lines). The plot of the initial ⁻OSCN formation rate versus the eriodictyol concentration (B) revealed an optimum flavonoid concentration of about 1 µM. At higher concentration the (pseudo-) halogenating LPO activity slightly decreased. A reciprocal re-plot of the data according to Lineweaver-Burk (C) yielded a linear dependence. From the intersection of the curve with the x- and y-axis K_m and V_max values of 0.358±0.005 µM and 0.262±0.003 µM/s, respectively, were obtained. Mean and standard deviation of n=3 experiments are given.

Fig. 3

K_m and V_max values of aromatic compounds. The shown enzymatic parameters correspond to benzoic acid derivatives (A), phenylethanoids (C) and cinnamic acid derivatives (E), respectively while in (B), (D) and (F) the corresponding basic chemical structures are shown. Regarding the enzyme-kinetic data the black circles represent the K_m values while the V_max values are shown as white squares. For all substance classes compounds with a 3,4-dihydroxyphenyl moiety emerged as the most efficient LPO activity regenerators. The given standard deviations correspondent to the R² value observed during the linear fit of the experimental data according to Lineweaver-Burk. Mean and standard deviation of n=3–9 experiments are given.

Fig. 4

K_m and V_max values of selected flavonoids. The enzymatic parameters shown in (A) again correspond to the K_m value (black circles) and the V_max value (white squares) of the flavonoids. In (B) the basic chemical structure of flavonoids is given and in (C) the chemical differences between the tested flavonoids are shown. Thereby the connection between C₂ and C₃ of the C ring is indicated as single bond (SB) or double bond (DB). All flavonoids showed a quite high affinity to LPO (low K_m value). Regarding the V_max value those compounds with a monohydroxylation at the B ring (naringenin and apigenin) exhibited a quite weak regenerating effect on the LPO activity. In contrast the flavonoids with a 3,4-dihydroxylated B ring emerged as excellent LPO substrates. Mean and standard deviation of n=3–5 experiments are given.

Fig. 5

Dependence of the enzymatic specificity constant from the distribution coefficient. Compounds with an 3,4-dihydroxy- (black), 3-hydroxy- (gray) or 4-hydroxy- (white) phenyl moiety were plotted against their logD value. Their affiliation to the different substance classes is also indicated. Among one hydroxylation pattern hydrophobic compounds (e.g. phenylethanoids) always showed stronger LPO activity-regenerating effects than hydrophilic ones (e.g. benzoic acid derivatives). Within one substance class the compounds with 3-4-dihydroxyphenyl moiety always had the strongest impact on the LPO-mediated ⁻OSCN production.

Fig. 6

Reaction rates of 3,4-dihydroxybenzoic acid with LPO. Compound I of the enzyme was pre-formed by incubating native LPO with a two-fold excess of H₂O₂ for 150 ms. Afterwards substrate was added and spectral changes were followed. While in (A) spectra were recorded after the pre-incubation of 2 µM LPO with 4 µM H₂O₂ in the absence of substrate in (B) 20 µM 3,4-dihydroxybenzoic acid were added. Spectra recorded 5 (black, bold), 66, 947 (bold), 1280, 1560, 1720, 2088, 2530, 4069, 6507 and 10,000 ms (gray, bold) after mixing are shown. In the presence of substrate (B) within the first second a quick transition from Compound I (bold black line, Soret band at 412 nm) to Compound II (bold gray line, Soret band to 431 nm, characteristic spectral changes around 550 nm) was observed which was considerably faster than in its absence (A). This reaction was followed by a slower backformation of native LPO (increase in the absorbance at 412 nm, isosbestic points at 423, 469 and 517 nm) within the next 9 s (bold light gray line) which was not observed in the negative control. For both transitions k_obs values were determined by following the increase (C) and decrease (E) of the absorbance at 431 nm. The data shown in (C and E) correspond to 1 µM LPO, 2 µM H₂O₂ and 20 µM 3,4-dihydroxybenzoic acid. By plotting the k_obs values against the substrate concentration (D and F) second order rate constants for the reaction of 3,4-dihydroxybenzoic acid with LPO Compound I (C, 8.09×10⁵ M⁻¹ s⁻¹) and Compound II (E, 2.90 ×10⁴ M⁻¹ s⁻¹) were determined. Mean and standard deviation of n=3 experiments are given.

Scheme 1

Perturbation of the (pseudo-)halogenating LPO activity. After the activation of native (ferric) LPO to Compound I by H₂O_2, the enzyme quickly oxidizes SCN⁻ to ⁻OSCN under the regeneration of native LPO. Yet especially in the presence of excess H₂O₂ this (pseudo-)halogenating activity is disturbed due to the formation of enzymatic redox intermediates which are not involved in ⁻OSCN production. This includes the spontaneous transition to Compound I* whose electronic structure at the heme center resembles Compound II. While Compound I* can lead to irreversible enzyme inhibition Compound II can further react with H₂O₂ to Compound III. The resolution of the latter by oxygen release yields the ferrous form of native LPO which is again quickly transformed to Compound II in the presence of H₂O₂. Spontaneous transitions are shown as thin arrows while standard arrows indicate reactions of LPO redox intermediates with H₂O₂ or SCN⁻. The reaction of LPO Compound I and II with aromatic compounds is shown as bold arrows. First and second order rate constants were taken from the literature , , .