Methylmercuric iodide modification of lipoxygenase-1. Effects on the anaerobic reaction and pigment bleaching

The effect of methylmercuric iodide modification of sulfhydryl groups in soybean lipoxygenase-1 on linoleate oxidation, carbonyl production and beta-carotene and chlorophyll alpha bleaching were determined under aerobic and anaerobic conditions. Linoleate oxidation at pH 9.0 was strongly inhibited by modification of the enzyme. On the other hand, pigment bleaching was enhanced with the modified enzyme. Unmodified lipoxygenase-1 was not sensitive to chlorophyll inhibition, but activity of modified lipoxygenase-1 was affected. Linoleate oxidation was inhibited up to 70% when 2.2 microM chlorophyll was present in the reaction mixture. Chlorophyll inhibition was similar with affinity chromatography-purified lipoxygenase-2 and modified lipoxygenase-1. Unmodified lipoxygenase-1 exhibited high bleaching activity under anaerobic conditions and relatively low activity under aerobic (oxygen or air) conditions. Modified lipoxygenase-1 showed a significant increase in carotene and chlorophyll bleaching under both anaerobic and aerobic conditions. Under anaerobic conditions in the presence of either pigment, both modified and unmodified lipoxygenase-1 exhibited high 285 nm absorbing material production. Antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, alpha-tocopherol, propyl gallate and tertiary butylated hydroxyquinone ) were powerful inhibitors of pigment bleaching by modified lipoxygenase-1. However, only tertiary butylated hydroxyquinone and propyl gallate blocked the increase in the rate of absorbance at 285 nm.     

Effect of modification of soybean lipoxygenase-1 with N-bromosuccinimide on linoleate oxidation,pigment bleaching and carbonyl production

Essential tryptophan residues were specifically modified in soybean lipoxygenase-1 by N-bromosuccinimide (NBS). Both linoleate oxidation and pigment bleaching (β-carotene or chlorophyll a) activities were significantly reduced with the modified enzyme under aerobic conditions. However, the effect on the reduction of linoleate oxidation was more marked. Pigment bleaching under anaerobic conditions was almost completely blocked with the modified enzyme. On the basis of spectral studies it was elucidated that soybean lipoxygenase-1 contains two essential tryptophan residues in its active site.     

A wavelength dependent mechanism for rose bengal-sensitized photoinhibition of red cell acetylcholinesterase

A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.     

Spin trapping study of reactive oxygen species formation during bopindolol peroxidation

The generation of singlet molecular oxygen ((1)O(2)) and hydroxyl radicals (HO*) during peroxidation of bopindolol in the presence of Co(II) ions was studied using electron spin resonance (ESR) and spectrophotometry methods. 2,2,6,6-Tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline-1-oxide were used as traps. The spectrophotometry determination of (1)O(2) was based on bleaching of p-nitrosodimethylaniline (RNO), which was caused by the product of the reaction of (1)O(2) with imidazole and was followed by monitoring the decrease in optical density at 440 nm. The effect of (1)O(2) quenchers and oxygen free radical scavengers on the ESR signal and the bleaching of RNO was studied. The data presented here give new evidence for generation of the reactive oxygen species during peroxidation of bopindolol.     

利用RNO脱色反应检测类囊体中的单线态氧

光敏剂RB在光照射下与O₂反应产生 ¹O₂, ¹O₂与组氨酸或咪唑反应的中间产物使RNO发生氧化,导致RNO在440 nm处吸光度减小,此即为RNO脱色反应.RNO脱色反应随着光照时间的增加而增大,表明RB受光照射后使 ¹O₂增加;随着组氨酸或咪唑浓度的增加,RNO脱色反应增大;咪唑在RNO脱色反应中的作用更明显. ¹O₂淬灭剂NaN₃或DABCO存在时,RNO脱色反应降低.利用RNO脱色反应检测到莴苣类囊体在强光照射下产生的 ¹O₂,随着光强和照射时间增加,类囊体中 ¹O₂的产生增加.     

Bleaching of Dichlorophenolindophenol by a Coupled Oxidation with Linoleate Catalyzed by Soybean Lipoxygenase

The coupled bleaching of 2,6-dichlorophenolindophenol by soybean lipoxygenase-1, was found to occur only under anaerobic conditions with a characteristic lag phase quite unlike the wellknown induction phase associated with lipoxygenase-catalyzed oxidation of linoleate hydroperoxide (LOOH)—free linolelic acid. The duration of this distinctive lag phase was very sensitive to lipoxygenase concentrations and equalled the length of time required for the primary enzyme activity to render the reaction solution virtually anaerobic. The onset of bleaching was marked by a gradual build-up of a ketodiene presumably derived from LOOH. Singlet O₂ and superoxide anion did not appear to be involved in the enzyme- catalyzed bleaching while the xanthine-xanthine oxidase system known to produce O₂^? was effective in bleaching DCPIP. It is proposed that the bleaching reaction was a result of ah oxidative and irreversible alteration of DCPIP involving a number of reactive oxidants known to be produced anaerobically upon incubation of LOOH and linoleic acid with native lipoxygenase.     

Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.     

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a K_i value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with K_in value of 27 μM and 24 μM, respectively, and k₃ value of 0.12 min⁻¹ and 0.35 min⁻¹, respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.     

Activity and stability of immobilised soybean lipoxygenase-1 in aqueous and supercritical carbon dioxide media

The activity of immobilised soybean lipoxygenase-1 (LOX-1) was studied in aqueous and supercritical carbon dioxide (SCCO₂) media for the production of 13S-hydroperoxyoctadecadenoic acid (13S-HPODE). In SCCO₂, it was optimal at 33 °C and 25 MPa. A higher space-time yield of 5.7×10^–3 Ms^–1 mg^–1 LOX-1 for 13S-HPODE was obtained in SCCO₂ compared to only 5×10^–5 Ms^–1 mg^–1 LOX-1 in aqueous medium. The stability of immobilised LOX-1 was only significantly affected by the pressurisation and depressurisation steps during reactions in SCCO₂.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (lambda>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (¹O₂) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of ¹O₂. In addition, the generation of ¹O₂ was verified further in the presence of D₂O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN₃). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of ¹O₂ by PBPs are as follows: APC > C-PC > R-PE.     

The anticancer activity of visnagin,isolated from Ammi visnaga L., against the human malignant melanoma cell lines,HT 144

Melanoma is a cancer of melanocyte cells and has the highest global incidence. There is a need to develop new drugs for the treatment of this deadly cancer, which is resistant to currently used treatment modalities. We investigated the anticancer activity of visnagin, a natural furanochromone derivative, isolated from Ammi visnaga L., against malignant melanoma (HT 144) cell lines. The singlet oxygen production capacity of visnagin was determined by the RNO bleaching method while cytotoxic activity by the MTT assay. Further, HT 144 cells treated with visnagin were also exposed to visible light (λ ≥ 400 nm) for 25 min to examine the illumination cytotoxic activity. The apoptosis was measured by flow cytometry with annexin V/PI dual staining technique. The effect of TNF-α secretion on apoptosis was also investigated. In standard MTT assay, visnagin (100 µg/mL) exhibited 80.93% inhibitory activity against HT 144 cancer cell lines, while in illuminated MTT assay at same concentration it showed lesser inhibitory activity (63.19%). Visnagin was induced apoptosis due to the intracellular generation of reactive oxygen species (ROS) and showed an apoptotic effect against HT 144 cell lines by 25.88%. However, it has no effect on TNF-α secretion. Our study indicates that visnagin can inhibit the proliferation of malignant melanoma, apparently by inducing the intracellular oxidative stress.

     

The effects of light-induced reduction of the photosystem II reaction center

Accumulation of reduced pheophytin a (Pheo-D1) in photosystem II reaction center (PSII RC) under illumination at low redox potential is accompanied by changes in absorbance and circular dichroism spectra. The temperature dependences of these spectral changes have the potential to distinguish between changes caused by the excitonic interaction and temperature-dependent processes. We observed a conformational change in the PSII RC protein part and changes in the spatial positions of the PSII RC pigments of the active D1 branch upon reduction of Pheo-D1 only in the case of high temperature (298 K) dynamics. The resulting absorption difference spectra of PSII RC models equilibrated at temperatures of 77 K and 298 K were highly consistent with our previous experiments in which light-induced bleaching of the PSII RC absorbance spectrum was observable only at 298 K. These results support our previous hypothesis that Pheo-D1 does not interact excitonically with the other chlorins of the PSII RC, since the reduced form of Pheo-D1 causes absorption spectra bleaching only due to temperature-dependent processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Site-directed mutations at D1-His198 and D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803: deciphering the spectral properties of the PSII reaction centre

Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll PD1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll ChlD1, was replaced with His. The D1-His198Gln mutation produces a 3nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the (P+QA--PQA) absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls PD1/PD2. The D1-Thr179His mutation produces a 2nm displacement to the red of the bleaching minimum in the Qy region of the (3P-1P) absorbance difference spectrum. Analysis of the flash-induced (P+QA--PQA) and (3P-1P) absorbance difference spectra of both mutants compared with wild-type at 80K indicate that the cation of the oxidized donor P+ is predominantly localized on the chlorophyll PD1 of the special pair and that the reaction centre triplet state, produced upon charge recombination from 3[P+Pheo-], when the primary quinone electron acceptor QA is doubly reduced, is primarily localized on ChlD1.     

Subpicosecond and picosecond studies of electron transfer intermediates in Rhodopseudomonas sphaeroides reaction centers

The primary electron transfer processes in isolated reaction centers of Rhodopseudomonas sphaeroides have been investigated with subpicosecond and picosecond spectroscopic techniques. Spectra and kinetics of the absorbance changes following excitation with 0.7-ps 610-nm pulses, absorbed predominantly by bacteriochlorophyll (BChl), indicate that the radical pair state P⁺BPh^?, in which an electron has been transferred from the BChl dimer (P) to a bacteriopheophytin (BPh), is formed with a time constant no greater than 4 ps. The initial absorbance changes also reveal an earlier state, which could be an excited singlet state, or a P⁺BChl^? radical pair.The bleaching at 870 nm produced by 7 ps excitation pulses at 530 nm (absorbed by BPh) or at 600 nm (absorbed predominantly by BChl) shows no resolvable delay with respect to standard compounds in solution, suggesting that the time for energy transfer from BPh to P is less than 7 ps. However, the bleaching in the BPh band at 545 nm following 7-ps 600-nm excitation, exhibits an 8- to 10-ps lag with respect to standard compounds. This finding is qualitatively similar to the 35-ps delay previously observed at 760 nm by Shuvalov at al. (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Matveetz, Y.A. and Kryukov, P.G. (1978) FEBS Lett. 91, 135–139) when 25-ps 880-nm excitation flashes were used. A delay in the bleaching approximately equal to the width of the excitation flash can be explained in terms of the opposing effects of bleaching due to the reduction of BPh, and absorbance increases due to short-lived excited states (probably of BChl) that turn over rapidly during the flash.The decay of the initial bleaching at 800 nm produced by 7-ps 530- or 600-nm excitation flashes shows a fast component with a 30-ps time constant, in addition to a slower component having the 200-ps kinetics expected for the decay of P⁺BPh^?. The dependence on excitation intensity of the absorbance changes due to the 30-ps component indicate that the quantum yield of the state responsible for this step is lower than that observed for the primary electron transfer reactions. This suggests that at least part of the transient bleaching at 800 nm is due to a secondary process, possibly caused by excitation with an excessive number of photons. If the 800-nm absorbing BChl (B) acts as an intermediate electron carrier in the primary photochemical reaction, electron transfer between B and the BPh must have a time constant no greater than 4 ps.     

Site-directed mutations at D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803 modify the spectroscopic properties of the accessory chlorophyll in the D1-branch of the reaction center

D1-Thr179, which overlies the reaction center chlorophyll Chl D1 of Photosystem II was replaced with His and Glu through site-directed mutation in Synechocystis sp. PCC 6803. Spectroscopic characterization of the mutants indicates that, compared to wild type, the main bleaching in the triplet-minus-singlet absorbance difference spectrum and the electrochromic band shift in the (P680 (+)Q A (-)-P680Q A) absorbance difference spectrum are displaced to the red by approximately 2 nm in the D1-Thr179His mutant and to the blue by approximately 1 nm in the D1-Thr179Glu mutant. These difference spectra are compared with the absorbance difference spectra, measured on the same states in the D1-His198Gln mutant in which the axial ligand D1-His198 of the special pair chlorophyll, P D1, was replaced by glutamine. Together, these results give direct evidence that (a) the reaction center triplet state, produced upon charge recombination from (3)[P (+)Pheo (-)], is primarily localized on Chl D1; (b) the cation of the oxidized donor P (+) is predominantly localized on chlorophyll P D1 of the special pair; and (c) the Q Y band of the accessory chlorophyll Chl D1 is electrochromically shifted in response to charges on P (+) and Q A (-). Light-induced absorbance difference spectra (between 650 and 710 nm), associated with the oxidation of secondary donors and the reduction of Q A, exhibit a bleaching attributed to the oxidation of a Chl Z and strong electrochromic band shifts. On the basis of mutation-induced spectroscopic changes and of structure-based calculations, we conclude that the experimental spectra are best explained by a blue-shift of the Q Y band of the accessory chlorophyll Chl D1, arising from charges on Car D2 (+) and Chl ZD2 (+) and on reduced Q A.     

Isoxaflutole treatment leads to reversible tissue bleaching and allows for more effective detection of GFP in transgenic soybean tissues

The effects of the herbicide isoxaflutole (IFT) on tissue growth and the detection of green fluorescent protein (GFP) in transgenic embryogenic soybean tissues were evaluated using image analysis. The inclusion of this “bleaching” herbicide at 3 or 10 mg l⁻¹ in a standard soybean embryo proliferation medium resulted in a change in tissue color from green to non-pigmented over the course of a 4-wk experiment. Although the loss in pigmentation was observed in transgenic and non-transformed control tissues, tissue growth remained unaffected. GFP expression in three different transgenic soybean clones, representing low to moderate GFP expression levels, was easily detected and quantified using image analysis following culture of the tissues on an IFT-containing medium. Quantification of GFP in tissues from the same clones cultured in the absence of IFT, however, was difficult using image analysis. After transfer of transgenic embryogenic tissue from a medium containing IFT to a medium without IFT, the growth of pigment-containing tissue resumed. The bleaching effects from this herbicide appear to be reversible and make IFT and possibly other bleaching herbicides useful in the analysis of GFP expression in tissues, where interference from chlorophyll is problematic.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Product yield in oxygenation of linoleate by soybean lipoxygenase: the value of the molar extinction coefficient in the spectrophotometric assay

Two iodimetric methods and a gravimetric method were used to determine the spectrophotometric molar absorptivity of the purified product of lipoxygenase-catalyzed dioxygenation of linoleate (13-LS-hydroperoxy-cis,trans-9,11-octadecadienoate). Earlier determinations had led to the use of values varying from 24,000 to 28,000 M-1 cm-1 for epsilon at 235 nm. In the current work, the two iodimetric values (spectrophotometric and titrimetric) average 22,500, while gravimetric analysis of scrupulously purified material gives 22,900. Final 235-nm absorbancies for lipoxygenase runs over a wide range of linoleic acid concentrations up to 200 microM give a constant final percentage completion. If one assumes a 100% reaction, epsilon is 23,600. Each method has less than 1.5% standard error; the average of the three independent methods is 23,000 +/- 580 (2.5%), all being lower than the previous values. In the enzyme-catalyzed reaction of linoleate at less than 200 microM substrate, only 235-nm-absorbing material is formed. Above 200 microM linoleate, yields at 235 nm decrease and yields of materials absorbing at 280 nm increase (the latter is known to arise from lipoxygenase-catalyzed reaction of linoleyl hydroperoxide). Below 200 microM substrate, linoleate purified by HPLC produces only one HPLC-observable product, 13-linoleyl hydroperoxide. At higher substrate concentrations other HPLC peaks arise, again with higher wave-length absorptions. Spectrophotometric data using the epsilon determined here agree with those from the O2 electrode. It is concluded that at S less than 200 microM, saturating air, and sufficient enzyme, soybean lipoxygenase-1 produces a sole product and the reaction proceeds to completion.     

Enzymatic cycling method using creatine kinase to measure creatine by real-time detection

We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP–GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP–GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay.