Prosthetic heme modification during halide ion oxidation. Demonstration of chloride oxidation by horseradish peroxidase

Myeloperoxidase (MPO), eosinophil peroxidase (EPO), and chloroperoxidase can oxidize iodide, bromide, and chloride, but most peroxidases, including the prototypical horseradish peroxidase (HRP), reportedly only oxidize iodide and, in some cases, bromide. We report here that incubation of HRP with Br(-) and H(2)O(2) at acidic pH results in both bromination of monochlorodimedone and modification of the heme group. Mass spectrometry indicates that the heme 2- and 4-vinyl groups are modified by either replacement of a vinyl hydrogen by a bromide or addition of HOBr to give a bromohydrin. These reactions do not occur if protein-free heme and Br(-) are co-incubated with H(2)O(2) or if the HRP reaction is carried out at pH 7. Surprisingly, similar prosthetic heme modifications occur in incubations of HRP with H(2)O(2) and Cl(-). A mechanism is proposed involving oxidation of Br(-) or Cl(-) to give HOBr or HOCl, respectively, followed by addition to a vinyl group. In the reaction with Cl(-), a meso-chloro heme adduct is also formed. This first demonstration of Cl(-) oxidation by HRP, and the finding that prosthetic heme modification occurs when Br(-) or Cl(-) is oxidized in the absence of a cosubstrate, show that only modest tuning is required to achieve the unique chloride oxidation activity of MPO and EPO. The results raise the question of how the prosthetic hemes of MPO and EPO, whose function is to produce oxidized halide species, escape modification.     

HPLC isolation and NMR structure elucidation of the most prominent octabromo isomer in technical octabromo diphenyl ether

The major octabromo isomer of technical octabromo diphenyl ether mixture (technical octaBDE) DE-79 was isolated by RP-HPLC. Three serially coupled columns (each 250 mm long) enabled a good separation of the target compound from other congeners using 100% ACN as eluent. Approximately 100 microg of the target compound was isolated with a purity of >90% and investigated by MS for confirmation of the degree of bromination. 1H-NMR and 2-D 1H-13C correlation NMR spectra unequivocally clarified that the octaBDE in question is 2,2',3,3',4,4',6,6'-octabromodiphenyl ether (BDE 197). Based on annual production rates of technical BDE products (1999/2001), approximately 380 tons of BDE 197 were annually produced which, on the long term, may enter the environment. Compared with other individual BDE congeners, BDE 197 has the seventh highest application rate. Reductive debromination of BDE 197 can lead to four hepta-, 15 hexa-, 23 penta-, and 28 tetra-BDE isomers, respectively. This variety includes all known major BDEs of environmental concern (BDE 47, 85, 99, 100, 153, 154, and 183). The identification of BDE 197 in technical octaBDE DE-79 strongly suggests that research on the environmental fate of BDEs should include this key-BDE congener.     

Abnormal solvent effects on hydrogen atom abstractions. 1. The reactions of phenols with 2,2-diphenyl-1-picrylhydrazyl (dpph*) in alcohols

Rate constants, k(ArOH/dpph*)(S), for hydrogen atom abstraction from 13 hindered and nonhindered phenols by the diphenylpicrylhydrazyl radical, dpph*, have been determined in n-heptane and a number of alcoholic and nonalcoholic, hydrogen-bond accepting solvents. Abnormally enhanced k(ArOH/dpph*)(S) values of have been observed in alcohols. It is proposed that this is due to partial ionization of the phenols and a very fast electron transfer from phenoxide anion to dpph*. The popular assessment of the antioxidant activities of phenols with dpph* in alcohol solvents will generally lead to an overestimation of their activities.     

Studies of reaction heats and structure of complexes formed between strong N-bases and phenols

The heats of reactions between various phenols and two strong N-bases of guanidine-like character in acetonitrile, are determined. The values can be used as a measure of self-assembly abilities of the phenol molecules in the interactions with strong N-bases, playing a very important role in biological systems. In the case of TBD complexes with corresponding nitrophenols, the protonated N-base is hydrogen-bonded to the nitro group excluding the self-assembly process of the phenols. In the case of other phenols, the self-assembly abilities are dependent on pK_a values of phenols. With increasing acidity of phenols their ability to form the hydrogen-bonded chains decreases. The maximum of length of the chains is observed for 4-methylphenol, which has a similar pK_a value to that in the tyrosine residue in biological systems.     

Synthesis of poly(alkylene phosphate)s with n-containing bases in the side chains. III. N1-Oxoethyleneuracil on the poly(trimethylene phosphate) chain

Poly(2-hydro-2-oxo-1,3,2-dioxaphosphorinane) was quantitatively chlorinated, and the resulting polymer was reacted with excess of imidazole, giving the highly reactive polyesteramide. This polymer, treated with N¹-hydroxyethyluracil, gave polyphosphates with N¹-oxoethyleneuracil in the side chains. The final polymers (M?_n ≈ 10⁴) as well as the intermediate products were characterized by ¹H-, ¹³C- and ³¹P-NMR spectroscopy.     

Isotope shift measurements in titanium I

The use of an effusive beam of titanium atoms crossed with a CW single-mode tunable dye laser has allowed the high-resolution, Doppler-free study of the isotope shifts between⁵⁰Ti,⁴⁸Ti and⁴⁶Ti, for seven 3d ² 4s ² a³ F _J 3d ² 4s 4p z ⁵ D _J , visible transitions of Ti I. The measurements show without ambiguity the existence of a non-negligible field shift. Using the values of the nuclear radii of titanium (coming from muonic X-ray measurements), it is possible to determine the respective values of the field and mass shifts.     

Determination of dipole moment in the ground and excited state by experimental and theoretical methods of N-nonyl acridine orange

The absorption and fluorescence spectra of N-nonyl acridine orange are determined at room temperature (298 K) in cyclohexane, benzene, carbon tetrachloride, chloroform, chlorobenzene and dichloromethane. The ground state of dipole moment was obtained by impedance measurements using Guggenheim-Debeye's method. The experimental excited state dipole moment of N-nonyl acridine orange was determined using Bakhshiev's and Kawski-Chamma-Viallet's formulae and solvent polarity parameter proposed by Reichardt. These experimental results were completed with theoretical results using quantum chemical methods. The experimental (muexp=10.76 D) and theoretical (mucal=9.9 D) dipole moments in the ground and excited state (muexp*=14.56 D) were compared.     

Autocatalytic modification of the prosthetic heme of horseradish but not lactoperoxidase by thiocyanate oxidation products. A role for heme-protein covalent cross-linking

The mammalian peroxidases eosinophil peroxidase, lactoperoxidase (LPO), and myeloperoxidase oxidize thiocyanate to the antimicrobial agents hypothiocyanous acid (HOSCN) and (SCN)2 and are part of a defense system that protects the host from infections. Horseradish peroxidase (HRP), a plant enzyme, also oxidizes thiocyanate. We report here that the prosthetic heme vinyl groups of HRP react with the catalytically generated HOSCN and (SCN)2 to form at least nine vinyl-modified heme adducts. Mass spectrometry combined with analysis of the equivalent reactions of HRP reconstituted with 2- or 4-cyclopropylheme, or mesoheme-d4, shows that all of the prosthetic heme modifications result from addition of oxidized thiocyanate to the heme vinyl groups. No delta-meso-substitution of the heme was observed, in contrast to what is observed with radical agents. Model studies show that incubation of either HRP with preformed HOSCN or a solution of heme with preformed (SCN)2 gives rise to the same products obtained in the HRP-catalyzed reaction. Model studies also demonstrate that the SCN* radical, if formed, should add to a meso-carbon. These findings implicate an electrophilic addition mechanism. In contrast, oxidation by LPO of thiocyanate, the normal substrate of this enzyme, does not result in heme modification. In view of the demonstrated intrinsic reactivity of the heme group, LPO must actively suppress heme modification. As the key difference between LPO (and other mammalian peroxidases) and HRP is the presence of two covalent ester links between the heme and the protein, we propose that these links contribute to steric protection of the adjacent heme vinyl groups.     

Acid-base reactions of 1,3,4-thiadiazolines and thiocarbonyl ylides; 1,3,4-thiadiazoline-2-spiro-2′-adamantane

The surprising formation of C₂₂H₃₂N₂S₂ from the title compound 1 at 45°C involves the interaction of the basic adamantanethione S-methylide ($\text{2}$) with its acidic precursor $\text{1}$, in the course of which the anion $\text{6}$ undergoes electrocyclic ring opening; the acid and base functions offer the clue to a prolific chemistry of the thiadiazoline $\text{1}$ and the thiocarbonyl ylide $\text{2}$.     

ContinuousK-theory

We study arithmetical properties of homotopy groups of thel-adic completion of Quillen'sK-theory space of number field, with a view on the Dwyer-Friedlander comparison map into étaleK-theory. The relation of these groups toK-theory is a complete analogy to the relation of continuous étale cohomology to étale cohomology. We identify the torsion subgroup of the resulting term with the subgroup of divisible elements inK _2n (F). We prove that this term is sent isomorphically into étaleK-theory, giving some further evidence for the Lichtenbaum-Quillen conjectures.