H. Plat

Bromoperoxidase from Ascophyllum nodosum: a novel class of enzymes containing vanadium as a prosthetic group?

Abstract Reconstitution experiments showed that the transition metal vanadium is essential for enzymic activity of bromoperoxidase from the marine brown alga Ascophyllum nodosum. A linear relationship existed between brominating activity and the amount of vanadium incorporated in the enzyme. Maximal activity was found when the enzyme contained 5.1 nmol vanadium per mg bromoperoxidase. Based on gel-exclusion high-performance liquid chromatography, the molecular mass of bromoperoxidase was estimated as 90 000. Chemical analysis and electron paramagnetic resonance (EPR) demonstrated that bromoperoxidase contains vanadium (4.4 nmol per mg bromoperoxidase). This value corresponds to a ratio of 0.4 mol vanadium per mol bromoperoxidase. The EPR experiments suggest that in bromoperoxidase the vanadium ion is present in the 5 + redox state. These experiments demonstrate for the first time the existence of an enzyme containing vanadium at the active site.

Isolation procedure and some properties of myeloperoxidase from human leucocytes

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.

Vanadium(V) as an essential element for haloperoxidase activity in marine brown algae: purification and characterization of a vanadium(V)-containing bromoperoxidase from Laminaria saccharina

Abstract The purification and characterization of bromoperoxidase from the marine brown alga Laminaria saccharina is described. The purified enzyme is homogeneous, as verified by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography; it has a dimeric structure with a relative molecular mass of 108 kDa. Reconstitution experiments showed that the transition metal vanadium is essential for the brominating activity of this enzyme. Analysis of bromoperoxidase with electron paramagnetic resonance in combination with activity measurements indicated that the ratio of vanadium ligated to the protein at the active site increased from 0.3 to 2.0 when the purified enzyme was incubated with an excess of the metal. Properties of bromoperoxidase from L. saccharina are compared with bromoperoxidase from Ascophyllum nodosum (De Boer, E., Van Kooyk, Y., Tromp, M.G.M., Plat, H. and Wever, R. (1986) Biochim. Biophys. Acta 869, 48–53). The presence of ‘vanadium(V)-dependent’ haloperoxidase activity in crude extracts of Fucus spiralis, F. serratus, F. vesiculosus, Pelvetia canaliculata and Chorda filum is described. These experiments demonstrate that vanadium-containing haloperoxidases are more widely spread in nature

The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions.

The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.

Some properties of human eosinophil peroxidase, a comparison with other peroxidases.

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.

Isolation procedure and some properties of the bromoperoxidase from the seaweed Ascophyllum nodosum

Abstract A large-scale isolation procedure for bromoperoxidase from the brown alga Aseophyllum nodosum is described. The purified enzyme showed one major band (40 kDa) after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The activity of the enzyme in the bromination of monochloridimedone was studied. The rate of the reaction showed optima between pH 4.5 and pH 6.5, the position of which was determined by the concentration of both Br− and H2O2. The enzyme was not inhibited by H2O2, azide or cyanide and only weakly by Br−. The Km for bromide of 12.7 mM is independent of pH (6.0–7.6). However, the Km for H2O2 increased with increasing H+ concentration. Bromoperoxidase was thermostable and was resistant to high concentrations of organic solvents such as methanol, ethanol and 1-propanol.

Human eosinophil peroxidase: a novel isolation procedure, spectral properties and chlorinating activity.

Like neutrophils, eosinophils contain a high concentration of a peroxidase that may have enzymic activity which is associated with the antiparasitic function of these cells [ 11. In neutrophils it has been demonstrated that the peroxidase (myeloperoxidase) is involved in the killing of micro-organisms (reviewed in [2]). This antimicrobial activity of myeloperoxidase is probably due to the ability of this enzyme to oxidize Clwith HzOz to the reactive hypochlorous acid (HOCl) [3-51. It is not known whether human eosinophil peroxidase can catalyse the same reaction. It was recently shown [6], however, that eosinophil peroxidase partially purified from guinea pig eosinophils is bactericidal when combined with Hz02 and Cl-. Studies on human eosinophil peroxidase are hampered since eosinophils can only be obtained in significant amounts from blood from patients with eosinophilia. However, in order to be able to purify the peroxidase from these eosinophils and to study the enzyme in some more detail, sufficient patient blood must be available, which is generally not the case. In addition, it is conceivable that the properties of patient eosinophils, and the enzymes in these cells, differ from those normally foundin blood circulation. As shown in [7] the amount of haem iron derived from the peroxidase in an eosinophil is -4-times higher than the concentration of myeloperoxidase in a neutrophil. Since white blood cells of normal donors contain 3-S% eosinophils, 12-20% of the total peroxidase (on the basis of haem iron) in a homogenate of white cells must be due to eosinophil peroxidase. From 100 1 human blood 200 mg pure myeloperoxidase can be obtained [8]; it should therefore be pos-

The bromoperoxidase from the red alga Ceramium rubrum also contains vanadium as a prosthetic group

Abstract A novel bromoperoxidase was isolated from the marine red alga Ceramium rubrum (Ceramiaceae, Rhodophyta); for the enzymic activity of this peroxidase the transition metal vanadium proved to be essential. The presence of vanadium in the enzyme preparation was established by atomic absorption spectrophotometry. Like the vanadium-containing bromoperoxidases from brown algae, this enzyme was not inhibited by cyanide or azide. This is the first report of the occurrence of a vanadium-containing bromoperoxidase in a red seaweed.

Purification and some characteristics of a non-haem bromoperoxidase from Streptomyces aureofaciens.

A bromoperoxidase was isolated from the chlortetracycline-producing actinomycete, Streptomyces aureofaciens. This enzyme catalysed bromination and iodination, but surprisingly did not catalyse chlorination. The enzyme had an acidic pH optimum (pH 4.3) and the isoelectric point was 3.5. The Km for bromide was 20 mM and the Km for H2O2 was as high as 8 mM. The bromoperoxidase did not contain haem, since it was not inhibited by azide or cyanide. Excess bromide or chloride had no effect on its brominating activity; however, fluoride strongly inhibited the bromoperoxidase (Ki = 20 microM). On the basis of gel electrophoresis in the absence and presence of sodium dodecyl sulphate, the molecular mass of the enzyme was 65 kDa and it consisted of two subunits of 32 kDa each. The bromoperoxidase was remarkably thermostable.

Vanadium ― an element involved in the biosynthesis of halogenated compounds and nitrogen fixation

In nature, and in particular in the marine environment, a wide variety of halometabolites is found. Marine algae, for example, are a rich source of bromometabolites [l]. Some of the algae, such as the brown seaweeds (Phaeophyta) Ascophyilum nodosum and Fucus vesiculosus, contribute enormous quantities of organo-halo species to our ecosystem and form an important source of bromine-containing material released to the atmosphere [2]. The role of these halogenated compounds is not always clear. Some appear to participate in biological defence mechanisms, since they show antimicrobial or antifeeding properties, others may act as chemical messengers in communication systems. A number of enzymes involved in the biosynthesis of these compounds have been purified and characterised, and it was up to now generally accepted that peroxidases containing haem are involved in the halogenation reaction. The mechanism by which halogenation occurs is still open to debate (discussed in the book by Neidleman and Geigert [ 11). Some authors suggest that the haloperoxidases generate free hypohalous acid which will react aspecifically with a broad range of nucleophilic acceptors, whereas