Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. The enzyme uses hydrogen peroxide to oxidize chloride (Cl-), bromide (Br-), iodide (I-), and the pseudohalide thiocyanate (SCN-) to their respective hypohalous acids. This study for the first time presents transient kinetic measurements of the oxidation of these halides and thiocyanate by the myeloperoxidase intermediate compound I, using the sequential mixing stopped-flow technique. At pH 7 and 15 degrees C, the two-electron reduction of compound I to the native enzyme by Cl- has a second-order rate constant of (2.5 +/- 0.3) x 10(4) M(-1) s(-1), whereas reduction of compound I by SCN- has a second-order rate constant of (9.6 +/- 0.5) x 10(6) M(-1) s(-1). Iodide [(7.2 +/- 0.7) x 10(6) M(-1) s(-1)] is shown to be a better electron donor for compound I than Br- [(1.1 +/- 0.1) x 10(6) M(-1) s(-1)]. The pH dependence studies suggest that compound I reduction by (pseudo-)halides is controlled by a residue with a pKa of about 4.6. The protonation of this group is necessary for optimum (pseudo-)halide anion oxidation. These transient kinetic results are underlined by steady-state spectral and kinetic investigations. SCN- is shown to be most effective in shifting the system myeloperoxidase/hydrogen peroxide from the peroxidatic cycle to the halogenation cycle, whereas iodide is shown to be more effective than bromide which in turn is much more effective than chloride. Decreasing pH increases the rate of this transition. Our results show that thiocyanate is an important substrate of myeloperoxidase in most environments and that hypothiocyanate is likely to contribute to leukocyte antimicrobial activity.

Hypericin is a naturally occurring substance found in the common St. John's 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-beta-hydroxylase, 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 (N-methyl-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 promising 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 promising 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 (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4 -8. At ؊1 at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK a of (4.3 ؎ 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.

Compound I of peroxidases takes part in both the peroxidation and the halogenation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human eosinophil peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-mixing stopped-flow technique. Addition of 1 equiv of hydrogen peroxide to native EPO leads to complete formation of compound I. At pH 7 and 15 degrees C, the apparent second-order rate constant is (4.3 +/- 0.4) x 10(7) M(-1) s(-1). The rate for compound I formation by hypochlorous acid is (5.6 +/- 0.7) x 10(7) M(-1) s(-1). EPO compound I is unstable and decays to a stable intermediate with a compound II-like spectrum. At pH 7, the two-electron reduction of compound I to the native enzyme by thiocyanate has a second-order rate constant of (1.0 +/- 0. 5) x 10(8) M(-1) s(-1). Iodide [(9.3 +/- 0.7) x 10(7) M(-1) s(-1)] is shown to be a better electron donor than bromide [(1.9 +/- 0.1) x 10(7) M(-1) s(-1)], whereas chloride oxidation by EPO compound I is extremely slow [(3.1 +/- 0.3) x 10(3) M(-1) s(-1)]. The pH dependence studies suggest that a protonated form of compound I is more competent in oxidizing the anions. The results are discussed in comparison with those of the homologous peroxidases myeloperoxidase and lactoperoxidase and with respect to the role of EPO in host defense and tissue injury.

Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono-and dithiols by the MPO intermediates, Compound I (k Q ) and Compound II (k R ), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39 þ 0.12U10 S M 3I s 3I and 9.16 þ 1.63U10 S M 3I s 3I ), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and K Klipoic acid showed k Q values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k Q dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants (k R ) of 1.33 þ 0.08U10 S M 3I s 3I , 5.25 þ 0.07U10 S M 3I s 3I and 3.03 þ 0.07U10 Q M 3I s 3I . Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27 þ 0.05U10 Q M 3I s 3I ), cysteine methyl ester (8.14 þ 0.08U10 Q M 3I s 3I ), cysteine ethyl ester (3.76 þ 0.17U10 Q M 3I s 3I ) and K K-lipoic acid (4.78 þ 0.07U10 R M 3I s 3I ) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.z 1999 Federation of European Biochemical Societies.