The importance of hydroperoxide activation for the detection and assay of mammalian 5-lipoxygenase

Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1-5 microM) during the reaction which effected a 10-20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.     

High-performance liquid chromatography with mercury cathode electrochemical detection: application to lipid hydroperoxide analysis

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were <0.3 pmol and <30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.     

Comments on recent reports on infrared spectral detection of disease markers in blood components

The search for disease markers in whole blood, or easily accessible blood components by spectral methods is a highly important aspect in the field of biophotonic research for disease diagnostics and screening, since it promises a minimally invasive approach to assess an individual's state of health. Fourier transform infrared spectroscopy, in particular, promises to be a fast, inexpensive method to search for markers of disease, since it detects variation in the proteome, lipidome and metabolome of biofluids, or activation of immune cells. However, the analysis of any materials by spectral methods is confounded by external factors such as those related to sample deposition and data acquisition, and by inherent variations in blood plasma concentration of small molecules (lactate, carbonate, phosphate, glucose) that varies between individual subjects and even for a given individual, as a function of time. Furthermore, observed differences in spectral patterns between patient samples and the control group may be due to the body's immune response (in particular, to the albumin to globulin ratio) and therefore, may not be specific to disease. These factors need to be accounted for in any effort to reliably detect much smaller variations in the concentration of disease‐specific markers.     

Lipoxygenase, hydroperoxide isomerase, and hydroperoxide cyclase in young cotton seedlings

Lipoxygenase was demonstrated in young cotton seedlings. It catalyzed the oxygenation of linoleic or linolenic acid, predominantly at carbon 13, and its molecular weight was estimated by gel filtration to be 100,000. Hydroperoxide isomerase was also present and converted hydroperoxylinoleic or hydroperoxylinolenic acid to α- or γ-ketols. The enzyme utilized the 13-hydroperoxy isomer in preference to the 9 isomer and its molecular weight was estimated at 250,000 by gel filtration. In addition, hydroperoxide cyclase, which catalyzes the conversion of 13-hydroperoxylinolenic acid to 12-oxo-phytodienoic acid, was present. Hydroperoxide isomerase and hydroperoxide cyclase activities could not be separated by gel filtration and ion-exchange chromatography experiments, indicating the two enzyme activities may be associated with the same protein. The activities of all three enzymes were very low in the seed but increased immediately after germination, reached a maximum after 3 to 4 days, and then declined. The results suggest a role, as yet unknown, for these enzymes during early plant development.     

Effect of lipid hydroperoxide on lipoxygenase kinetics.

In order to investigate the activation of lipoxygenase and to clarify the role of the oxygenation product hydroperoxide in this process, the effect of 13-hydroperoxylinoleic acid (P, 0-35 microM) on linoleic acid (S, 1-80 microM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 microM O2 with rapid kinetic techniques. The following observations were made: (1) Iron(II) and iron(III) lipoxygenases are kinetically different: reactions started with the Fe(II) enzyme form show a lag phase, whereas iron(III) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [S] greater than 50 microM, the initial burst in the iron(III) lipoxygenase curves is still followed by a lag. The lag phase disappears completely only in the presence of micromolar quantities of P. (3) The approximate dissociation constants for S and P are 15 and 24 microM, respectively, 1 order of magnitude smaller than the corresponding values in the absence of oxygen. The observed kinetics are predicted by numerical integration of the rate equations of a model based on the single lipid binding site mechanism for the anaerobic lipoxygenase reaction [Ludwig et al. (1987) Eur. J. Biochem. 168, 325-337; Verhagen et al. (1978) Biochim. Biophys. Acta 529, 369-379]. A quasi-steady-state approximation of the model suggests that a high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.     

Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

AIM: To identify alkyl hydroperoxide reductase subunit C(AhpC) homologs in Bacillus subtilis(B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.METHODS: Two AhpC homologs(AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues(C37S, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.     

Intracellular distribution of hydroperoxide isomerase

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Sporicidal activity of tertiary butyl hydroperoxide

Tertiary butyl hydroperoxide (t-BOOH) was found to be sporicidal for Bacillus megaterium ATCC19213. Sporicidal action was very temperature dependent, and the potency of t-BOOH increased about tenfold for each increase in temperature of 15 °C over the range from 30° to 70 °C. At still higher temperatures, heat and molar levels of t-BOOH were mutually potentiating for killing. Vegetative cells and germinated spores were some thousand times less resistant to t-BOOH than dormant spores. The order of resistance for spores was: Bacillus stearothermophilus ATCC7953 > Bacillus subtilis var. niger = Bacillus megaterium ATCC33729 > Bacillus megaterium ATCC19213. Killing was not enhanced by decoating and occurred without germination or loss of refractility of the spores. Spore resistance to t-BOOH was lower at more acid pH values and was decreased also by demineralization. Spores could be protected by the chelator o-phenanthroline, especially in association with Fe²⁺. Overall, it seemed that killing was associated with nonmetabolic formation of alkyl peroxyl radicals, which are thought to be responsible for killing of vegetative cells by organic hydroperoxides.Abbreviation A-BOOH tertiary butyl hydroperoxide     

Lipid hydroperoxide levels in plant tissues

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and are key intermediates in the octadecanoid signalling pathway in plants. Lipid hydroperoxides (LHPO) were determined spectrophotometrically based on their reaction with an excess of Fe(2+)at low pH in the presence of the dye xylenol orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in Phaseolus: microsomes, senescing potato leaves and in a range of other plant tissues including Phaseolus hypocotyls (26+/-5 nmol g(-1) FW), Alstroemeria floral tissues (sepals 66+/-13 nmol g(-1) FW petals 49+/-6 nmol g(-1) FW), potato leaves (334+/-75 nmol g(-1) FW), broccoli florets (568+/-68 nmol g(-1) FW) and Chlamydomonas cells (602+/-40 nmol g(-1) FW). Relative to the total fatty acid content of the tissues, the % LHPO was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. In order to relate the levels of LHPO to specific signalling pathways, transgenic potato plant lines were used in which lipoxygenase (LOX) (responsible for hydroperoxide biosynthesis) and hydroperoxide lyase (a route of hydroperoxide degradation) activities were largely reduced by an antisense-mediated approach. While the LHPO levels were similar to wild type in the individual LOX antisensed plants, basal LHPO levels, by contrast, were elevated by 38% in transgenic potato leaves antisensed in hydroperoxide lyase, indicating a role for this enzyme in the maintenance of cellular levels of LHPOs.     

Sterol hydroperoxide metabolism by Salmonella typhimurium

In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.