Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

Hemin-catalyzed generation of triplet acetone

Hemin can substitute for horseradish peroxidase as a catalyst for the aerobic oxidation of isobutanal to acetone and formate. Previous studies have shown that the chemiphosphorescent emission observed in the enzyme-catalyzed reaction is due to the production of acetone in its triplet state. Although no chemiphosphorescence is observed with the model system (hemin), generation of triplet acetone in this system is indicated by an analysis of data for energy transfer to the 9,10-dibromoanthracene-2-sulfonate ion and for interception of the excited species by the sorbate ion, a known triplet quencher. These data are compared to those obtained with triplet acetone generated by thermal cleavage of tetramethyldioxetane in aqueous solution. The results are in agreement with the hypothesis that the quenching of triplet acetone by oxygen is less efficient in the enzyme catalyzed reaction, pointing to a protective role for the apo-enzyme in that system.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Weak chemiluminescence of bilirubin and its stimulation by aldehydes

Bilirubin in an alkaline solution exhibits a weak chemiluminescence (CL) under aerobic conditions. This spontaneous CL was markedly enhanced by the addition of various aldehydes. The fluorescent emission spectrum of bilirubin, excited by weak intensity light at 350 nm, coincided with its CL emission spectrum (peak at 670 nm). CL emission from bilirubin was not quenched by active oxygen scavengers. This suggests that triplet oxygen reacts with bilirubin, and forms an oxygenated intermediate (hydroperoxide) as a primary emitter (oxidative scission of tetrapyrrole bonds in bilirubin is not involved in this CL). The Ehrlich reaction (test for monopyrroles) and hydrolsulphite reaction (test for dipyroles) on the CL reaction mixture and unreacted bilirubin showed no differences. When the CL was initiated by singlet oxygen, rather than superoxide anion, monopyrrole, was detected in the reaction products by gel chromatography. The inhibitory effect of a scavenger of singlet oxygen on CL was eliminated in the presence of formaldehyde. Therefore, triplet carbonyl, formed by singlet oxygen through the dioxetane structure in bilirubin, is not an emitter. The reaction mechanism of bilirubin CL and the formation of a hydroperoxide intermediate is discussed in relation to the chemical structure of luciferin molecules from bioluminescent organisms.     

The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits.

1. A particulate enzyme fraction and an acetone powder preparation from cucumber fruits cleaved 9- and 13-hydroperoxyoctadecadienoic acids to form volatile aldehydes and oxoacid fragments. 2. From the 9-hydroperoxide, the major volatile fragments were cis-3-nonenal and trans-2-nonenal using particulate enzyme and acetone powder preparations, respectively. 3. Hexanal was the only significant volatile fragment from the 13-hydroperoxide. 4. The particulate enzyme system was equally effective on both 9- and 13-hydroperoxide isomers and was fully active under anaerobic conditions and at pH 6.4. 5. An enzymic pathway for the biogenesis of hexanal, cis-3- and trans-2-nonenal (components of the characteristic flavour volatiles of cucumber) from linoleic acid is proposed. This involves the sequential activity of lipoxygenase, hydroperoxide cleavage and cis-3-: trans-2-enal isomerase enzymes.     

Purification of lipoxygenase and hydroperoxide dehydrase in flaxseeds: interaction between these enzymatic activities.

We have purified two enzymic activities from flaxseed acetone powder: a lipoxygenase and a hydroperoxide dehydrase. The lipoxygenase activity belongs to an iron-containing protein having a molecular weight of 130 kDa which, upon incubation with alpha-linolenic acid, forms 13-hydroperoxy-9(Z), 11(E), 15(Z)- octadecatrienoic acid. The hydroperoxide dehydrase (a 55 kDa protein) metabolizes this hydroperoxide to an allene oxide which in turn is spontaneously hydrolyzed to alpha- and gamma-ketols. Relationships between these two enzymes were studied and results suggest an inhibition of the lipoxygenase by hydroperoxide dehydrase.     

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.     

Photochemical oxidation of chlorpromazine in the dark induced by enzymically generated triplet carbonyl compounds

Enzymically generated triplet acetone and ethanal transfer energy to chlorpromazine as indicated by (i) suppression of the acetone chemiphosphorescence (ii) concomitant formation of chlo$\text{r}$ promazine photoproducts, that is the radical cation and the sulfoxide (iii) inhibition of photoproduct formation by a very efficient competition for triplet carbonyl energy using the sodium salt of 9,10-dibromoanthracene-2-sulfonic acid.This is the first report of a photooxidation in the dark.     

Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.     

Interaction between enzyme-generated triplet carbonyls and molecules intercalated into DNA

The phosphorescence from enzyme-generated and -protected triplet acetone is very efficiently quenched by dyes intercalated into DNA. The process is unlikely to be due to energy transfer and is tentatively ascribed to electron transfer occurring within the DNA helix complex with the acting enzyme. This quenching markedly protects DNA from breaks induced by triplet acetone. In the case of some barely emissive enzyme-generated triplet carbonyl species, it is possible to detect a weak emission resulting from the interaction with dye X DNA; this emission may be associated with back electron transfer.     

Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study

Nanosecond laser flash photolysis and pulse radiolysis were used to generate and characterize the triplet state and cation radical of C-phycocyanin (C-PC) from Spirulina platensis. The transient absorption spectra of C-PC were measured from direct excitation and acetone sensitization in aqueous solution at room temperature by KrF (248 nm) laser flash photolysis. Laser-induced transient species have been characterized by the method of acetone sensitization and one-electron oxidation. In nitrous oxide-saturated phosphate buffer saline (pH = 7.0) of C-PC, the produced intermediates are assigned to the excited triplet state and the radical cation. Using acetone as photosensitizer, the C-PC excited triplet states produced via triplet-triplet energy transfer and the C-PC radical cation from electron transfer reaction were further confirmed. Furthermore, the corresponding kinetic parameters were determined. To our knowledge, the transient absorption spectra of C-PC have been reported for the first time.     

Damages induced in lambda phage DNA by enzyme-generated triplet acetone

Exposure of lambda phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

Photochemical synthesis of substituted indan-1-ones related to donepezil.

A photoenolization reaction is shown to be the key reaction step in the preparation of substituted indan-1-ones as convenient precursors for the synthesis of donepezil, a well-known acetylcholinesterase inhibitor. Model 2,5-dialkylphenacyl chlorides, differently substituted in the alpha-carbon position, were found to produce indan-1-ones upon irradiation in non-nucleophilic solvents in high chemical yields via hydrogen chloride release. While direct excitation of 4,5-dimethoxy-2-methylphenacyl chloride led to a complex mixture of photoproducts, photolysis of the corresponding benzoate was found to form 5,6-dimethoxyindan-1-one in 62-72% chemical yields and a relatively low quantum efficiency (Phi approximately 0.02). This compound can then be easily converted to donepezil by standard synthetic steps described in the literature. Isotopic exchange and quenching experiments revealed that the product is obtained by the photoenolization process via the triplet excited state, while minor side-photoproducts originate from the singlet excited state. Irradiation of the reactant in neat acetone, used both as a triplet sensitizer and solvent at the same time, was found to form 5,6-dimethoxyindan-1-one exclusively in high (90%) chemical yield.     

Acetone enhancement of cumene hydroperoxide supported microsomal aniline hydroxylation.

Acetone was found to enhance both cumene hydroperoxide and NADPH/oxygen supported microsomal aniline hydroxylation. Since cumene hydroperoxide supported aniline hydroxylation does not involve an electron transport system, these results suggest that acetone produces its enhancing effect by facilitating either the formation of an activated oxygen species or the insertion of oxygen into the substrate.     

Damages induced in λ phage DNA by enzyme-generated triplet acetone

Exposure of λ phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus leteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

Studies on the properties of the singlet oxygen-like factor produced during lipid peroxidation

The singlet oxygen reaction product of various trapping agents is observed during enzymic and nonenzymic peroxidation of microsomes as well as during the peroxidation of pure lipids extracted from microsomes. We now wish to report that purified fatty acid hydroperoxide alone, as well as peroxidized microsomal lipid and cumene hydroperoxide also form the singlet oxygen reaction product with 2,5-diphenylfuran. The reaction product (cis-1,2-dibenzoylethylene) was observed to be formed in an anaerobic system, with or without EDTA. The data indicate that a reaction of hydroxyl radicals with 2,5-diphenylfuran cannot account for the formation of dibenzoylethylene in these systems. These results are consistent with a hypothesis that the singlet oxygen-like factor was formed from the lipid peroxides per se and, in addition, supports the possibility that either the peroxides can react directly with diphenylfuran to produce dibenzoylethylene or that the self-reaction of organic peroxides may form an intermediate product which can react directly with singlet oxygen-trapping agents to produce substances which are identical to a reaction of the trapping agents with singlets oxygen.     

Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the O? O bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H₂O₂ by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl-hydroxo complexes, as that formed during the oxidation of metmyoglobin by H₂O₂, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.     

Identification of carbon-centred radicals derived from linalyl hydroperoxide, a strong skin sensitizer: a possible route for protein modifications

Few studies are reported on the formation of reactive carbon-centred radical species from toxic xenobiotics. In this paper the formation of carbon radicals derived from the skin sensitizer linalyl hydroperoxide is described using radical trapping and EPR studies. Radical trapping used TMIO as scavenger agent and light, heat or TPP-Fe(3+) as radical inducers. EPR spin trapping was based on the use of the parent alcohol, generating the same allyloxyl radical than the hydroperoxide by photolysis of the corresponding nitrite formed with t-BuONO, also playing the role of the spin trap. It is suggested that the generation of these carbon radical species could play an important role for the binding of the hydroperoxide with skin proteins to form antigenic structures, the first step of the skin sensitization mechanism.     

Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase

Linoleic acid oxidation catalyzed by lipoxygenase (lipoxidase) activity in extracts of defatted corn germ does not terminate in the product, linoleic acid hydroperoxide, unless the lipoxygenase is first partially purified. If purification is not attempted, the hydroperoxide product exists only as a barely detectable intermediate in the synthesis of three products. One of these was identified as 9-hydroxy-10-oxo-cis-12-octadecenoic acid formed from the hydroperoxide by the enzyme, linoleate hydroperoxide isomerase. Another product, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, is believed to be formed by an isomerase also. The third product was the linoleate ester of one of the hydroxy-oxo-fatty acids, 9-(cis-9,cis-12-octadecadienoyl)-10-oxo-cis-12-octadecenoic acid. It is not known if the synthesis of the ester is enzyme-catalyzed. When a mixture of 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid and 9-hydroperoxy-trans-10,cis-12-octa-decadienoic acid from soybean lipoxygenase oxidation of linoleic acid was used as a substrate, 13-hydroxy-12-oxo-cis-9-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid were formed as the major products of catalysis by linoleate hydroperoxide isomerase(s) from corn. Smaller quantities of 9-hydroxy-10-oxo-cis-12-octadecenoic acid and 13-hydroxy-10-oxo-trans-11-octadecenoic acid were also formed.     

Quenching of the triplet state of Safranine-O by aliphatic amines in AOT reverse micelles studied by transient absorption spectroscopy

The photophysics of Safranine-O (3,6-diamino-2,7-dimethyl-5 phenyl phenazinium chloride) (SfH(+)Cl(-)) was investigated in reverse micelles (RMs) of AOT (sodium bis(2-ethylhexyl)sulfosuccinate) with special emphasis on the triplet state processes. The triplet is formed in its monoprotonated form, independently of the pH of the water used to prepare the RMs. While the intersystem crossing quantum yields in RMs are similar to those in organic solvents, the triplet lifetime is much longer. Since the pH in the water pool of AOT RMs is close to 5 and the triplet state of the dye is subjected to proton quenching, the long lifetime indicates that the dye resides in a region where it cannot be reached by protons during its lifetime. All the measurements indicate that the dye is localized in the interface, sensing a medium of micropolarity similar to EtOH : water (3:1) mixtures. The quenching by aliphatic amines was also investigated. While the quenching by the hydrophobic tributylamine is similar to that in methanol, the hydro-soluble triethanolamine is one order of magnitude more effective in RMs than in homogeneous solution. In the latter case the quenching process is interpreted by a very fast intramicellar quenching, the overall kinetics being controlled by the exchange of amine molecules between RMs. Semireduced dye is formed in the quenching process in RMs in the di-protonated state with a comparable quantum yield to the monoprotonated state formed in homogeneous solvents. The results point to the advantage of the reverse micellar system for the generation of active radicals for the initiation of vinyl polymerization, since a much lower concentration of amine can be employed with similar quantum yields.