Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease.

Purpose: To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA.

Methods: Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay.

Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin.

Peroxynitrite, a highly reactive biological oxidant, is formed under pathophysiologic conditions from the diffusion-limited reaction of nitric oxide and superoxide radical anion. Peroxynitrite has been implicated as the mediator of nitric oxide toxicity in many diseases and as an important signaling disrupting molecule (L. Liaudet et al.

Ocular surface extracellular DNA and nuclease activity imbalance: a new paradigm for inflammation in dry eye disease.

Purpose: We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation.

Methods: Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva.

Cytochrome P450 119 Compounds I Formed by Chemical Oxidation and Photooxidation Are the Same Species.

Compound I from cytochrome P450 119 prepared by the photooxidation method involving peroxynitrite oxidation of the resting enzyme to Compound II followed by photooxidation to Compound I was compared to Compound I generated by m-chloroperoxybenzoic acid (MCPBA) oxidation of the resting enzyme. The two methods gave the same UV/Visible spectra, the same products from oxidations of lauric acid and palmitic acid and their (ω-2,ω-2,ω-3,ω-3)-tetradeuterated analogues, and the same kinetics for oxidations of lauric acid and caprylic acid. The experimental identities between the transients produced by the two methods leave no doubt that the same Compound I species is formed by the two methods.

Rates of fatty acid oxidations by P450 compound I are pH dependent.

The rates of oxidation of fatty acids by CYP119 compound I were dependent on the pH of the medium. The plot shows log k values for reactions of acids as a function of pH, where the slopes indicate mixed third-order and fourth-order dependence on base concentration. For palmitic acid, the rate increased 50-fold over the pH range 6.

Oxidation of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient.

Oxidations of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient were studied. The only product formed in Compound I oxidations was 10,11-epoxyundecanoic acid, whereas the enzyme under turnover conditions gave the epoxide and 9-hydroxy-10-undecenoic acid in a 10 : 90 ratio. Kinetic studies at 0 °C of oxidations by Compounds I formed by MCPBA oxidation and by a photo-oxidation pathway gave the same results, displaying saturation kinetics that yielded equilibrium binding constants and first-order oxidation rate constants that were experimentally indistinguishable.

Rate constants for cyclizations of α-hydroxy radical clocks.

The 1-hydroxy-1-methyl-6,6-diphenyl-5-hexenyl radical (4a) and the 1-hydroxy-1-methyl-7,7-diphenyl-6-heptenyl radical (4b) were prepared from the corresponding PTOC esters (anhydrides of a carboxylic acid and N-hydroxypyridine-2-thione). The key step in the synthetic method for the precursors was a coupling reaction of the respective carboxylic acids with the thiohydroxamic acid, which was conducted for ca. 5 min and followed rapidly by chromatography.

Low temperature photo-oxidation of chloroperoxidase Compound II.

Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as -50° C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene.

Kinetics and activation parameters for oxidations of styrene by Compounds I from the cytochrome P450(BM-3) (CYP102A1) heme domain and from CYP119.

Cytochrome P450 (CYP or P450) enzymes are ubiquitous in nature where they catalyze a vast array of oxidation reactions. The active oxidants in P450s have long been assumed to be iron(IV)-oxo porphyrin radical cations termed Compounds I, but P450 Compounds I have proven to be difficult to prepare. The recent development of an entry to these transients by photo-oxidation of the corresponding iron(IV)-oxo neutral porphyrin species (Compounds II) permits spectroscopic and kinetic studies.

Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.

Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially nonexistent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers.

Kinetics of oxidation of benzphetamine by compounds I of cytochrome P450 2B4 and its mutants.

Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite.

Highly reactive porphyrin-iron-oxo derivatives produced by photolyses of metastable porphyrin-iron(IV) diperchlorates.

Photolyses of metastable porphyrin-iron(IV) diperchlorates in laser flash photolysis reactions gave highly reactive transients. The systems studied were 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetramesitylporphyrin (TMP), and 2,3,7,8,12,13,17,18-octaethylporphyrin (OEP). The new species, which decayed within milliseconds in acetonitrile solutions, were shown to react with organic substrates by oxo-transfer reactions involving insertions into carbon-carbon double bonds of alkenes and styrenes or benzylic carbon-hydrogen bonds of arenes.

Spectra and kinetic studies of the compound I derivative of cytochrome P450 119.

The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting enzyme with peroxynitrite. The UV-vis spectrum of the Compound I species contained an asymmetric Soret band that could be resolved into overlapping transitions centered at approximately 367 and approximately 416 nm and a Q band with lambda(max) approximately 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxidized (alkene oxidation) and hydroxylated (C-H oxidation) products, as demonstrated by product studies and oxygen-18 labeling studies.

Tunneling in C-H oxidation reactions by an oxoiron(IV) porphyrin radical cation: direct measurements of very large H/D kinetic isotope effects.

Rate constants for oxidations of benzyl alcohol-d0 and -d7 by oxoiron(IV) tetramesitylporphyrin radical cation perchlorate in acetonitrile were measured in single turnover kinetic studies. The kinetic isotope effect (kH/kD) increased from 28 at 23 degrees C to 360 at -30 degrees C due to extensive hydrogen atom tunneling that was analyzed in terms of a parabolic energy barrier to tunneling. Similarly, large KIE values were found for oxidations of ethylbenzene-d0 and -d10 at room temperature.

Rate constants for 1,5- and 1,6-hydrogen atom transfer reactions of mono-, di-, and tri-aryl-substituted donors, models for hydrogen atom transfers in polyunsaturated fatty acid radicals.

Rate constants for 1,5- and 1,6-hydrogen atom transfer reactions in models of polyunsaturated fatty acid radicals were measured via laser flash photolysis methods. Photolyses of PTOC (pyridine-2-thioneoxycarbonyl) ester derivatives of carboxylic acids gave primary alkyl radicals that reacted by 1,5-hydrogen transfer from mono-, di-, and tri-aryl-substituted positions or 1,6-hydrogen transfer from di- and tri-aryl-substituted positions to give UV-detectable products. Rate constants for reactions in acetonitrile at room temperature ranged from 1 x 10(4) to 4 x 10(6) s(-1).

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative.

The cytochrome P450 enzyme CYP119, its compound II derivative, and its nitrosyl complex were studied by iron K-edge x-ray absorption spectroscopy. The compound II derivative was prepared by reaction of the resting enzyme with peroxynitrite and had a lifetime of approximately 10 s at 23 degrees C. The CYP119 nitrosyl complex was prepared by reaction of the enzyme with nitrogen monoxide gas or with a nitrosyl donor and was stable at 23 degrees C for hours.

Kinetic studies of a fast, reversible alkene radical cation cyclization reaction.

The radical cation formed by mesylate heterolysis from the 1,1-dimethyl-7,7-diphenyl-2-mesyloxy-6-heptenyl radical was studied in several solvents. Computational results suggest that the initially formed acyclic radical cation is a resonance hybrid with partial positive charge in both double bonds of 1,1-diphenyl-7-methyl-1,6-octadiene (10). Thiophenol trapping was used as the competing reaction for kinetic determinations.

Radical heterolysis reactions. Dynamics of formation, collapse, and solvation of ion pairs determined by a competition kinetic method.

The kinetics of radical heterolysis reactions, including rate constants for radical cation-anion contact ion pair formation, collapse of the contact pair back to the parent radical, and separation of the contact pair to a solvent-separated ion pair or free ions were obtained in several solvents for a beta-mesyloxy radical. Rate constants were determined from indirect kinetic studies using thiophenol as both a radical trapping agent via H-atom transfer and an alkene radical cation trapping agent via electron transfer. [reaction: see text].

Cytochrome p450 compound I.

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate.

Laser flash photolysis generation and kinetic studies of porphyrin-manganese-oxo intermediates. Rate constants for oxidations effected by porphyrin-Mn(V)-oxo species and apparent disproportionation equilibrium constants for porphyrin-Mn(IV)-oxo species.

Porphyrin-manganese(V)-oxo and porphyrin-manganese(IV)-oxo species were produced in organic solvents by laser flash photolysis (LFP) of the corresponding porphyrin-manganese(III) perchlorate and chlorate complexes, respectively, permitting direct kinetic studies. The porphyrin systems studied were 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPFPP), and 5,10,15,20-tetrakis(4-methylpyridinium)porphyrin (TMPyP). The order of reactivity for (porphyrin)Mn(V)(O) derivatives in self-decay reactions in acetonitrile and in oxidations of substrates was (TPFPP) > (TMPyP) > (TPP).

Formation of compound I by photo-oxidation of compound II.

[reaction: see text] Compound I is the heme-iron(IV)-oxo porphyrin radical cation formed in peroxidase and catalase enzymes by reaction with hydrogen peroxide. As an alternative to chemical oxidations of porphyrin-iron(III) species, various compound I species were produced by 355 nm laser flash photolysis photooxidation of the corresponding compound II species, porphyrin-iron(IV)-oxo derivatives. The method is demonstrated by production and kinetic studies of the compound I species from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin-iron, from horseradish peroxidase, and from wild-type horse skeletal myoglobin.

Kinetics of radical heterolysis reactions forming alkene radical cations.

Rate constants for heterolytic fragmentation of beta-(ester)alkyl radicals were determined by a combination of direct laser flash photolysis studies and indirect kinetic studies. The 1,1-dimethyl-2-mesyloxyhexyl radical (4a) fragments in acetonitrile at ambient temperature with a rate constant of k(het) > 5 x 10(9) s(-1) to give the radical cation from 2-methyl-2-heptene (6), which reacts with acetonitrile with a pseudo-first-order rate constant of k = 1 x 10(6) s(-1) and is trapped by methanol in acetonitrile in a reversible reaction. The 1,1-dimethyl-2-(diphenylphosphatoxy)hexyl radical (4b) heterolyzes in acetonitrile to give radical cation 6 in an ion pair with a rate constant of k(het) = 4 x 10(6) s(-1), and the ion pair collapses with a rate constant of k < or = 1 x 10(9) s(-1).

Solvent polarity effects and limited acid catalysis in rearrangements of model radicals for the methylmalonyl-CoA mutase- and isobutyryl-CoA mutase-catalyzed isomerization reactions.

The kinetics of reactions of models for the intermediate radicals formed in the methylmalonyl-CoA mutase- and isobutyryl-CoA mutase-catalyzed rearrangements were studied by laser flash photolysis methods. The aldehyde-containing model analogous to the propanal-3-yl radical reacted via 3-exo cyclization with rate constants that varied with solvent polarity (k in the range 2 x 105 to 1 x 107 s-1). The analogous methyl ketone-containing radical reacted 2 orders of magnitude less rapidly, and the ethylthiocarbonyl-containing radical analogue reacted too slowly for kinetic measurements.

Direct detection of ion pair formation and collapse in a migration reaction of a beta-phosphate radical.

In solutions of trifluorotoluene or toluene containing 2,2,2-trifluoroethanol, the beta-phosphate radical heterolyzed to give a detectable ion pair, identified as a solvent-separated species. Rate constants for the radical fragmentation reaction forming the ion pair, for ion pair collapse, and for diffusive escape to free ions were measured. The kinetics and entropy of activation for fragmentation indicate that the rearrangement reaction occurs by a heterolysis pathway in all solvents.