Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.     

Antioxidants suitable for use with chemiluminescence to identify oxyradical species

From a panel of 24 alleged antioxidants the most suitable antioxidants (AO) for use with chemiluminescence (CL) experiments were determined. Superoxide dismutase (SOD), using luminol as the chemiluminescence probe (Lum-CL), was inhibitory only towards O2*- and not HO* or (1)O2. SOD was thus a suitable antioxidant for O2*-, as was tiron. Tiron had advantages, however, since SOD acted as a pro-oxidant in the presence of H2O2 or H2O2/HO* generators. The two most suitable antioxidants for (1)O2 were diphenylisobenzofuran (DBF) and tryptophan, for both Lum and Lucigenin-CL (Luc-CL). Desferrioxamine, with both Lum and Luc-CL, was a very effective scavenger for HO*, but appeared to be an even more effective scavenger for (1)O2. Cysteamine showed the best discrimination between IC50s when the two (1)O2 generators NaOCl/H2O2 and NDPO2 were compared. Cysteamine was, therefore, the only scavenger that was appropriate for studies with hypochlorite. Melatonin, with Lum-CL, was found to be the most suitable scavenger for HO*. Mannitol, the classical AO for HO*, was not suitable when used with CL since it acted as a pro-oxidant. Some of the AOs revealed either calyx- or bell-shaped CL inhibition profiles and presumably, therefore, may act as both pro- or antioxidants at different concentrations. Antioxidants showing these kinds of dual activities should be used with caution in CL studies.     

On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.     

Oxidation of glutathione and superoxide generation by inorganic and organic selenium compounds

The carcinostatic activities of selenium (Se) compounds have been shown to be composition and concentration dependent. Several studies have indicated that the ratios between glutathione (GSH) and Se may play an important role in Se catalysis and toxicity. The present study examined the catalytic effect of three selenium compounds on GSH oxidation using lucigenin-dependent chemiluminescence (CL) as an indirect measure of superoxide generation. Various GSH:Se ratios were assayed for the glutathione oxidase activity of selenite, selenocystamine and diselenodipropionic acid. CL emitted from the reaction of selenite with GSH increased more rapidly and was greater than those from the diselenides, but the diselenide CL reactions were sustainable. Both selenite- and diselenide-induced CL were markedly suppressed by superoxide dismutase (SOD). Iodoacetic acid (IAc) effectively inhibited CL generated from selenite-, selenocystamine- and diselenodipropionic acid-catalyzed GSH oxidation. These results suggest that GSH oxidation catalyzed by selenite, and the diselenides selenocystamine and diselenodipropionic acid, generated the superoxide radical in which the CL was inhibited by SOD. Furthermore, CL inhibition by IAc suggests that the catalytic species producing superoxide were the GSSe(-) or RSe(-) anion. This redox chemistry may be responsible for selenite and organoselenium toxicity and apoptosis, making possible the design and synthesis of organoselenium-containing pharmaceuticals.     

Functional states of neutrophils as suggested by whole blood chemiluminescence

Luminol-enhanced chemiluminescence (CL) of whole blood was examined in order to distinguish between activation states of phagocytic cells. The CL response of these cells was provoked by a phagocytic stimulus--polystyrene particles. Four functional states of phagocytes were proposed: "resting", "stand by", "activated" and "exhausted". The distinction was done on the basis of extent of the CL response to the particles, time pattern of the process, inhibition of CL by plasma and appearance of spontaneous light emission. Freshly drawn blood of healthy individuals exhibits the "resting" profile of CL, but that of patients with bacterial infection reveals CL patterns ascribed in this paper to the "stand by", "activated" or "exhausted" states of phagocytes. The "stand by", "activated" and "exhausted" behaviour of phagocytes in extravasated blood may be induced by preincubation of blood, stimulation with saline extract of Escherichia coli or N-formyl-Met-Leu-Phe, and by some manipulations involved in preparation of the purified neutrophils.     

Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.     

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

Quantum dots‐based chemiluminescence probes: an overview

This mini‐review describes the recent developments in quantum dots‐based nanoprobes in liquid‐phase chemiluminescence (CL) analysis. In the referenced reports, multiple quantum dots (QDs) were adopted as final emission species either after direct oxidation reactions (direct CL) or after chemiluminescence resonance energy transfer (indirect CL). This review does not include papers in which QDs have been used as enhancers, catalysts, carriers or quenchers in chemiluminescence systems. A brief overview on the CL mechanisms of various QDs‐based nanoprobes and their analytical applications over the last decade is given, followed by comments on the future challenges and prospects in this field.     

Evidence for a novel Chlorella virus-encoded alginate lyase

To clone the genes encoding lysis protein from a Chlorella virus, water samples were collected from 13 aquatic environments located in the Kanto area of Japan. Eight water samples contained plaque-forming viruses on Chlorella sp. NC64A, but no virus was detected in the other five samples. A novel Chlorella virus, CVN1, was isolated from the Inba-numa marsh sample. CVN1 genomic DNA was partially digested and shotgun cloned into pUC118 to identify the genomic region responsible for the lytic phenotype on Chlorella sp. NC64A. A DNA fragment which encoded two ORFs, ORF1 and ORF2, was obtained by antialgal assay. The ORF2 gene product, CL2, consisted of 333 amino acids showing antialgal activity not only on the original host of Chlorella sp. NC64A, but also on the heterogeneous hosts of Chlorella vulgaris C-27 and C. vulgaris C-207. CL2 showed a weak homology (19.8% amino acid identity) to mannuronate lyase SP2 from Turbo cornutus. CL2 in Escherichia coli cells was purified using a nickel chelate column. Lyase activity of purified CL2 on alginic acid was observed in an enzyme assay. The specific activity of purified CL2 was 2.1x10(-2) U mg(-1), the optimum pH for enzymatic activity was 10.5, and Ca(2+) was required for enzyme activity. This is the first report of a Chlorella virus protein with lyase activity.     

Comparison of the interaction of tricyclic antidepressants with human polymorphonuclear leukocytes as monitored by the generation of chemiluminescence.

The interation of imipramine with human polymorphonuclear leukocytes (PMNs) results in a chemiluminescence (CL) response which has been attributed to the electronic excitation of the imipramine molecule resulting from a reaction of the drug with reactive oxygen species. In order to determine what portion of the tricyclic molecule is involved in this reaction, the interaction of other tricyclics with PMNs was monitored by chemiluminescence. It was observed that tricyclic antidepressants having a carbon atom at position 5 of the ring moiety (amitriptyline, for example) did not yield CL with either resting or zymosan-activated PMNs. In fact this group of compounds inhibited the zymosan-induced CL response. However, CL was observed, with both resting and metabolically-activated PMNs, from several tricyclics having a heterocyclic nitrogen at position 5. These included imipramine, desipramine, opipramol and iprindole. Chlorimipramine, which has a chlorine atom at position 3 of the ring system, failed to yield CL with resting or stimulated cells. Similarly, imipramine N-oxide failed to yield CL with resting cells, but enhanced CL was observed with zymosan-activated PMNs. On the basis of these observations it appears that some aspect of the ring moiety, other than just a heterocyclic nitrogen, facilitates a reaction between these molecules and reactive oxygen which culminates in the generation of CL.     

The ODN probes conjugating the Cu(II) complex enhance the luminol chemiluminescence by assembling on the DNA template

Potent peroxidase-like activity of the β-ketoenamine (1)-dicopper (II) complex (2) for the chemiluminescence (CL) of luminol either in the presence or absence of H(2)O(2) has been previously demonstrated by our group. In this study, the β-ketoenamine (1) as the ligand unit for copper(II) was incorporated into the oligonucleotide (ODN) probes. It has been shown that the catalytic activity of the ODN probes conjugating the ligand-Cu(II) complex is activated by hybridization with the target DNA with the complementary sequence. Thus, this study has successfully demonstrated the basic concept for the sensitive detection of nucleic acids by CL based on the template-inductive activation of the catalytic unit for CL.     

Two Processes Responsible for Chemiluminescence Development in the Course of Iron-Mediated Lipid Peroxidation

The kinetics of chemiluminescence (CL) accompanying Fe²⁺-induced lipid peroxidation (LPO) in liposome suspension has been investigated. A sequence of stages was observed, namely: (1) fast CL flash (FF); (2) latent period (LP); (3) slow CL flash (SF) and (4) stationary chemiluminescence (SL). The first three stages are known to reflect the Fe²⁺-mediated LPO process. In spite of the fact that at the stage of SL Fe²⁺ has completely oxidized and MDA has not accumulated, CL intensity was found to increase and after 0.5–1 h reached a value that was several times higher than SF amplitude. The maximal SL level was linearly dependent on the initial Fe²⁺ concentration and was not dependent on liposome concentration in the suspension. The nature of the processes responsible for CL emission at the stage of SL has been investigated using free radical reaction inhibitors and measurement of CL spectra. The SL spectrum was observed in the red region (λ>590 nm) in contrast to the SF CL spectrum (maximum at 540 nm). SL amplitude was strongly inhibited by sodium azide (40%), superoxide dismutase (SOD) (30%), desferrioxamine and EDTA (30%), whereas mannitol, ethanol, α-tocopherol and butylated hydroxytoluene were ineffective. The data obtained indicate that CL at the stage of SL is not directly related to LPO process, i.e. lipid free radical recombination. The mechanism of stationary CL generation is discussed.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.     

Inhibition of chemiluminescent response of olive flounder Paralichthys olivaceus phagocytes by the scuticociliate parasite Uronema marinum

Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian parasite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (ROIs) generated by respiratory bursts of phagocytes using zymosan as a stimulant. Cytotoxic and antioxidative activities of excretory-secretory (ES) products of the parasite were evaluated as well. Live U. marinum and its ES products had a negative and dose-dependent effect on luminol-enhanced CL responses of zymosan-stimulated phagocytes of olive flounder. After CL assay, the number of phagocytes showing viability was significantly reduced in the cells incubated with live U. marinum at ratios of 2:1 and 1:1 phagocytes:ciliates or ES products with 0.3 mg protein ml(-1) compared to controls. Lysis of phagocytes by exposure to ES products was observed also. ES products from U. marinum showed considerably high activities of superoxide dismutase (SOD) and catalase. The results of this study suggest that U. marinum can protect itself against host's phagocytes mediated oxidative damage by destroying phagocytes and scavenging ROIs.     

Anti-My-26: a monoclonal antibody inhibiting human phagocyte chemiluminescence

Anti-My-26, a mouse monoclonal IgG1 antibody, was raised against human granulocytes and has been shown to inhibit luminol-enhanced, glucose-independent chemiluminescence (CL) of human granulocytes (or monocytes) responding to the soluble secretagogues A23187 or ionomycin (calcium ionophores) and phorbol myristate acetate (PMA). Anti-My-26 inhibition of CL was reversible and was dependent on both secretatogue and monoclonal antibody concentration. This inhibition appeared to be directed at the component of granulocyte CL that is independent of NAD(P)H-oxidase-catalyzed formation of superoxide anion, because neither opsonized zymosan-stimulated CL nor the PMA-induced decrease in NAD (P)H-associated autofluorescence was affected by anti-My-26. In addition, ionomycin, over a wide concentration range, failed to generate any decrease in granulocyte autofluorescence. The A23187-induced CL inhibited by anti-My-26 was correlated with its depression of oxygen consumption. Furthermore, anti-My-26 was not cytotoxic and did not itself induce oxidative metabolism when used as a stimulant. Binding of anti-My-26 to phagocytic cells was not decreased by pre-exposure of cells to either A23187 or PMA. Evidence is presented to suggest that the binding of anti-My-26 to the granulocyte surface inhibits the oxidative response to calcium ionophore and PMA by blocking a common pathway(s) stimulated by these different secretagogues.     

Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 mumol AMP . h-1 . (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94 000 and 70 000. The Mr-70 000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5'-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration.     

Three-peaked chemilluminescent response of human peripheral blood leukocytes following stimulation with phytohaemagglutinin (PHA)

Luminol-enhanced chemiluminescence (CL) was used to examine the response of various leukocyte populations following stimulation with a crude extract of Phaseolus vulgaris, namely phytohaemagglutinin (PHA-C). Populations stimulated included a human peripheral mixed leukocyte preparation (MLP), and purified preparations of lymphocytes, monocytes and polymorphonuclear leukocytes (PMNL). Mouse peritoneal exudate cells and the lymphocytic cells lines Molt #4 and Daudi were also stimulated. Following stimulation, a characteristic three-peaked chemiluminescent response was obtained from the MLP population. Little or no response was obtained from the purified lymphocytes. Monocytes produced a sharp peak corresponding to the second peak of the MLP response and PMNL produced a broad peak corresponding to the third peak of the MLP response. Mouse peritoneal exudate cells containing lymphocytes and monocytes/macrophages showed a two-peaked stimulation which corresponded to the first two peaks of the MLP response. Molt #4 and Daudi showed no chemiluminescence if stimulated individually, but if added to a MLP substantial enhancement of the first and second peaks was observed. These results indicate some form of lymphocyte/monocyte interaction leading to enhanced CL following PHA-C stimulation.     

Inhibition and enhancement by organic compounds of luminol-KIO4-H2O2 chemiluminescence.

The effects of 36 organic compounds on luminol-KIO(4)-H(2)O(2) chemiluminescence (CL) were studied. It was found that most of the tested compounds could inhibit or enhance the CL intensity. The activities of such inhibitors or enhancers were related to the pH of the CL system and the number and position of functional groups such as -OH and -NH(2) on aromatic rings. The mechanism of the CL inhibition and enhancement was considered. Based on the CL inhibition or enhancement, the possibility of analytical applications was explored. The results demonstrated that numerous compounds were detectable at the ng/mL level using the CL system.     

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.     

Discovering severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays

An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CL(pro)) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small molecular compounds against SPECS database by virtual screening; 256 compounds were subsequently selected for biological evaluation. Through SPR technology-based assay, 52 from these 256 compounds were discovered to show binding to SARS-CoV 3CL(pro). The enzymatic inhibition activities of these 52 SARS-CoV 3CL(pro) binders were further applied to FRET-based assay, and IC(50) values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CL(pro) inhibitors were discovered. The fact that the obtained IC(50) values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (K(D)s) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CL(pro) inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.