Microfluidic peroxidase biochip for polyphenol synthesis

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.     

Soybean peroxidase as an effective bromination catalyst*

Soybean peroxidase (SBP), an acidic peroxidase isolated from the seed coat, has been shown to be an effective catalyst for the oxidation of a variety of organic compounds. In the present study, we demonstrate that SBP can catalyze halogenation reactions. In the presence of H(2)O(2), SBP catalyzed the oxidation of bromide and iodide but not chloride. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) served as a useful substrate for SBP-catalyzed halogenations yielding the 6-bromo derivative. Halogenation of veratryl alcohol was optimal at pHs below 2.5 with rates of 2.4 μm/min, achieving complete conversions of 150-μm veratryl alcohol in 24 h. The enzyme showed essentially no brominating activity at pHs above 5.5. SBP-catalyzed bromination of veratryl alcohol proceeded with a maximum reaction velocity, (V(max))(apparent), of 5.8 x 10(-1) μm/min, a K(m) of 78 μm and a catalytic efficiency (k(cat)/K(m) of 1.37 x 10(5) M/min at pH 4.0, assuming all of the enzyme's active sites participate in the reaction. SBP also catalyzed the bromination of several other organic substrates including pyrazole to produce a single product 1-bromopyrazole, indole to yield both 5-bromoindole and 5-hydroxyindole, and the decarboxylative bromination of 3,4 dimethoxy-trans-cinnamic acid to trans-2-bromo-1-(3,4 dimethoxyphenyl)ethylene. A catalytic mechanism for SBP-catalyzed bromination has been proposed based on experimental results in this and related studies.     

Chip-based polyketide biosynthesis and functionalization

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.     

Formation of DNA adducts in HL-60 cells treated with the toluene metabolite p-cresol: a potential biomarker for toluene exposure

We have examined DNA adduct formation in myeloperoxidase containing HL-60 cells treated with the toluene metabolite p-cresol. Treatment of HL-60 cells with the combination of p-cresol and H(2)O(2) produced four DNA adducts 1: (75.0%), 2: (9.1%), 3: (7.0%) and 4: (8.8%) and adduct levels ranging from 0.3 to 33.6 x 10(-7). The levels of DNA adducts formed by p-cresol were dependent on concentrations of p-cresol, H(2)O(2) and treatment time. In vitro incubation of p-cresol with myeloperoxidase and H(2)O(2) produced three DNA adducts 1: (40.5%), 2: (28.4%) and 3: (29.7%) with a relative adduct level of 0.7x10(-7). The quinone methide derivative of p-cresol (PCQM) was prepared by Ag(I)O oxidation. Reaction of calf thymus DNA with PCQM produced four adducts 1: (18.5%), 2: (36.4%), 3: (29.0%) and 5: (16.0%) with a relative adduct level 1.6x10(-7). Rechromatography analyses indicates that DNA adducts 1-3 formed in HL-60 cells treated with p-cresol and after myeloperoxidase activation of p-cresol were similar to those formed by reaction of DNA with PCQM. This observation suggests that p-cresol is activated to a quinone methide intermediate in each of these activation systems. Taken together, these results suggest PCQM is the reactive intermediate leading to the formation of DNA adducts in HL-60 cells treated with p-cresol. Furthermore, the DNA adducts formed by PCQM may provide a biomarker to assess occupational exposure to toluene.     

Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene

Cholesterol oxidase from Nocardia sp. was modified with a synthetic copolymer of polyoxyethylene allylmethyldiether (PEG) and maleic acid anhydride (MA anhydride), poly(PEG-MA anhydride). The modified cholesterol oxidase, in which 64% of the amino groups in the protein molecule were coupled to poly(PEG-MA), was soluble in organic solvents and catalyzed the oxidation reaction of cholesterol in benzene to form 4-cholesten-3-one with the enzymic activity of 0.6 mumol/min/mg protein. Using the modified cholesterol oxidase together with polyethylene glycol-modified peroxidase, coupled reactions shown below took place in Cholesterol + O2----4-Cholesten-3-one + H2O2 H2O2 + o-Phenylenediamine----H2O + Oxidized o-Phenylenediamine transparent benzene solution, not in an emulsified system. The oxidation of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phenylenediamine at 490 nm.     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.     

Thin-film IrOx pH microelectrode for microfluidic-based microsystems

Microsensors are valuable tools to monitor cell metabolism in cell culture volumes. The present research describes the fabrication and characterization of on-chip thin-film iridium oxide pH microsensors with dimensions of 20 microm x 20 microm and 20 microm x 40 microm suitable to be incorporated into nl volumes. IrOx thin films were formed on platinum microelectrodes by electrochemical deposition in galvanostatic mode. Anodically grown iridium oxide films showed a near super-Nernstian response with a slope of -77.6+/-2 mV/pH at 22 degrees C, and linear responses within the pH range of 4-11. Freshly deposited electrodes showed response times as low as 6s. Long-term studies showed a baseline drift of 2-3 mV/month, which could easily be compensated by calibration. This work demonstrated for the first time the use of planar IrOx pH microelectrodes to measure the acidification rate of CHO and fibroblast cells in an on chip cell culture volume of 25 nl with microfluidic control.     

Hydrogen peroxide biosensor based on direct electrochemistry of soybean peroxidase immobilized on single-walled carbon nanohorn modified electrode

Single-walled carbon nanohorns (SWCNHs) were used as a novel and biocompatible matrix for fabricating biosensing devices. The direct immobilization of acid-stable and thermostable soybean peroxidase (SBP) on SWCNH modified electrode surface can realize the direct electrochemistry of enzyme. Cyclic voltammogram of the adsorbed SBP displays a pair of redox peaks with a formal potential of -0.24 V in pH 5 phosphate buffer solution. The formal potential has a linear relationship with pH from 3 to 9 with a slope of -48.7 mV/pH, close to the value of -55.7 mV/pH expected at 18 degrees C for the reversible transfer of one proton and one electron. Bioactivity of SBP remains good in SWCNH microenvironment, along with effective catalysis of the reduction of H(2)O(2). In the absence of a mediator, this H(2)O(2) biosensor exhibited a high sensitivity (16.625 microAL/mmol), a linear range from 0.02 to 1.2 mmolL(-1), and a detection limit of 5.0 x 10(-7) mmolL(-1), as well as acceptable preparation reproducibility and excellent stability.     

Integration of microfluidics with a four-channel integrated optical Young interferometer immunosensor

This report describes an optical sensing hybrid system obtained by bonding a microfluidic system to an integrated optical (IO) four-channel Young interferometer (YI) chip. The microfluidic system implemented into a glass plate consists of four microchannels with cross-sectional dimensions of 200 microm x 15 microm. The microfluidic system is structured in such a way that after bonding to the IO chip, each microchannel addresses one sensing window in the four-channel YI sensor. Experimental tests show that the implementation of the microfluidics reduces the response time of the sensor from 100s, as achieved with a bulky cuvette, to 4s. Monitoring the anti-human serum albumine/human serum albumine (alpha-HSA/HSA) immunoreaction demonstrates the feasibility to use the microfluidic sensing system for immunosensing applications. In this case, a better discrimination between the bulk refractive index change and the layer formation can be made, resulting into higher accuracy and offering the prospect of being able to use the kinetics of the immunoreaction. The microfluidic sensing system shows an average phase resolution of 7 x 10(-5) x 2pi for different pairs of channels, which at the given interaction length of 4 mm corresponds to a refractive index resolution of 6 x 10(-8), being equivalent to a protein mass coverage resolution of 20 fg/mm2.     

Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos

After in vitro maturation and fertilization of porcine oocytes, the fertilized embryos were cultured under 5 or 20% oxygen (O2) for 7 days. In embryos cultured under 5% O2 versus 20% O2, development to the blastocyst stage was higher (36.3% versus 22.5%, P < 0.05); the hydrogen peroxide (H2O2) content as a reactive oxygen species was lower (92 pixels versus 111 pixels, P < 0.05); and fragmentation of DNA in 8- to 16-cell stage embryos (estimated by the comet assay) resulted in a shorter (P < 0.05) DNA tail (36 microm versus 141 microm). Antioxidants such as beta-mercaptoethanol (beta-ME) and Vitamin-E (Vit-E) suppressed oxidative damage in the embryos and improved their developmental ability. For embryos cultured under 20% O2, there were the following differences (P < 0.05) between embryos exposed to 0 microM versus 50 microM beta-ME: 28% versus 57% developed to the blastocyst stage; 125 pixels versus 98 pixels per embryo in H2O2 content; and a DNA tail of 209 microm versus 105 microm. In addition, for embryos cultured under 20% O2, there were also differences (P < 0.05) between those exposed to 0 microM versus 50 microM of Vit-E: 28% versus 40% rate of development to the blastocyst stage; 28.9 cells versus 35.9 cells in the expanded blastocyst; 122 pixels versus 95 pixels per embryo (H2O2 content); and 215 microm versus 97 microm length of the DNA tail. Therefore, a low O2 concentration during in vitro culture of porcine embryos decreased the H2O2 content and, as a consequence, reduced DNA fragmentation, and, thereby, improved developmental ability.     

Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols

A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of -50 mV versus Ag/AgCl without the requirement for addition of extraneous H(2)O(2), thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive "in-field" determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p-cresol.     

Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.     

The oxidative metabolism of thioglycollate-elicited mouse peritoneal macrophages: the relationship between oxygen, superoxide and hydrogen peroxide and the effect of monolayer formation

The oxygen and glucose metabolism of peritoneal macrophages harvested from untreated mice (resident cells) and mice given an i.p. injection of thioglycollate broth (thioglycollate cells) were examined. Thioglycollate cells consumed approximately 3 times as much O2 at rest and during phagocytosis as resident cells, but oxygen reduction products (superoxide and hydrogen peroxide) could be recovered in only minimal amounts despite triggering by phagocytosis or exposure to PMA. Indirect evidence for the formation of oxygen reduction products such as O2- by thioglycollate cells was obtained by observation of the major pathways for glucose oxidation and NBT dye reduction. When thioglycollate cells were allowed to adhere to a glass surface O2- and H2O2 were easily recovered in the extracellular medium with a 20-fold increase above cells in suspension exposed to PMA. This study suggests that thioglycollate-elicited macrophages have a vigorous oxidative metabolism but that recovery, and perhaps utilization, of O2 reduction products formed will depend on the conditions of incubation. These events may be significant both for the study of parameters of macrophage "activation" in vitro as well as the function of these cells in vivo.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV)

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..     

Covalent attachment of ferrocene to soybean peroxidase glycans: electron transfer mediation to redox enzymes

The electrochemical properties of native soybean peroxidase (SBP) and of SBP modified with covalently attached ferrocene electron-transfer mediators within microcavities etched at the tip of 25 mum radius platinum microelectrodes are reported. The microcavities incorporate approximately 50 fmol of SBP. In the microcavity, native SBP undergoes a relatively fast reduction but a very slow oxidation. Ferrocene mediators have been attached to the SBP glycans (Fc-SBP) (approximately 1.5 ferrocene mediators per SBP molecule). Cyclic voltammetry reveals that these centers are capable of mediating the reduction of oxidized SBP and increase the rate of heterogeneous electron transfer between the enzyme and the electrode by >10-fold. Micromolar concentrations of H2O2 chemically oxidize the SBP and Fc-SBP systems leading to an electrocatalytic reduction at approximately -0.1 V. Successive additions of 2.5 micromol of H2O2 at a constant applied potential of -0.1 V gave a steady-state constant current of approximately 60 nA within 20 s. The steady-state current increased linearly with peroxide concentration for 2.5 相似文献   

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.