Search

Hypericin is a naturally occurring substance found in the common St. Johns Wort (Hypericum species) and can also be synthesized from the anthraquinone derivative emodin. As the main component of Hypericum perforatum, it has traditionally been used throughout the history of folk medicine. In the last three decades, hypericin has also become the subject of intensive biochemical research and is proving to be a multifunctional agent in drug and medicinal applications. Recent studies report antidepressive, antineoplastic, antitumor and antiviral (human immunodeficiency and hepatitis C virus) activities of hypericin intriguing information even if confirmation of data is incomplete and mechanisms of these activities still remain largely unexplained. In other contemporary studies, screening hypericin for inhibitory effects on various pharmaceutically important enzymes such as MAO (monoaminoxidase), PKC (protein kinase C), dopamine-betahydroxylase, reverse transcriptase, telomerase and CYP (cytochrome P450), has yielded results supporting therapeutic potential. Research of hypericin and its effect on GABA-activated (gamma amino butyric acid) currents and NMDA (Nmethyl- D-aspartat) receptors also indicate the therapeutic potential of this substance whereby new insights in stroke research (apoplexy) are expected. Also in the relatively newly established fields of medical photochemistry and photobiology, intensive research reveals hypericin to be a promizing novel therapeutic and diagnostic agent in treatment and detection of cancer (photodynamic activation of free radical production). Hypericin is not new to the research community, but it is achieving a new and promizing status as an effective agent in medical diagnostic and therapeutic applications. New, although controversial data, over the recent years dictate further research, re-evaluation and discussion of this substance. Our up-to-date summary of hypericin, its activities and potentials, is aimed to contribute to this process.

Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free‐radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient‐state and steady‐state investigation of the reaction of 15 aliphatic and aromatic mono‐ and dithiols with horseradish peroxidase (HRP). Both sequential‐stopped‐flow spectrophotometric investigations of the reaction of HRP intermediates Compound I (k2) and Compound II (k3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2‐mercaptopurine) were excellent electron donors of both Compounds (k2: 104–107M−1s−1and k3: 103–106M−1s−1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates (k3<10 M−1s−1) for HRP (e.g. homocysteine, glutathione) and/or have a pKa,SH>9.5 (e.g. N‐acetylcysteine, α‐lipoic acid) were also shown to depend on exogenously added H2O2to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k3between 11 and 1600 M−1s−1(e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a pKa,SH<8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free-radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient-state and steady-state investigation of the reaction of 15 aliphatic and aromatic mono- and dithiols with horseradish peroxidase (HRP). Both sequential-stopped-flow spectrophotometric investigations of the reaction of HRP intermediates Compound I ( k2) and Compound II ( k3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2-mercaptopurine) were excellent electron donors of both Compounds ( k2: 10 4–10 7M −1s −1and k3: 10 3–10 6M −1s −1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates ( k3<10 M −1s −1) for HRP (e.g. homocysteine, glutathione) and/or have a p Ka,SH>9.5 (e.g. N-acetylcysteine, α-lipoic acid) were also shown to depend on exogenously added H 2O 2to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k3between 11 and 1600 M −1s −1(e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a p Ka,SH<8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

Cytosolic extracts of the cyanobacteriumAnacystis nidulansexhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Kmfor H2O2of 4.3 mM, a calculated turnover number of 7200 s−1, and an overall-rate constant of 3.5 × 106M−1s−1. The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki= 27.2 μM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 × 104M−1s−1at pH 7.0 and 25°C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.