Chemiluminescent acridan phosphate labelling compounds for detection in gels.

We have developed a chemiluminescent acridan phosphate labelling compound which produces a flash of light upon chemical triggering. Sequential treatment of the label with acid and a strong base, one of which contains a peroxide, initiates light emission as a rapid flash lasting 1--2 s. Labelling of analytes with these compounds permits their direct detection in a non-enzymatic assay format. Representative compounds have been linked to BSA as a model protein analyte and detected by chemiluminescence assay. Light intensity correlated with the amount of BSA over six orders of magnitude, permitting the detection of 50 amol of protein in solution. We have also demonstrated for the first time that labelled proteins can be separated by electrophoresis without destruction of the label and that chemiluminescence can be produced and detected directly in the gel. The ease, speed and sensitivity of detection by this new method should enable the development of simpler assays for a wide variety of analytes.

Electrochemiluminescence determination of 2',6'-difluorophenyl 10-methylacridan-9-carboxylate.

Electrochemical oxidation of the acridan 2',6'-difluorophenyl 10-methylacridan-9-carboxylate produces the corresponding acridinium ester, which reacts with hydrogen peroxide forming a dioxetanone intermediate. Decomposition of the dioxetanone generates light at 430 nm when it relaxes to the ground state. The effect of pH and hydrogen peroxide concentration on this ECL reaction and on the stability of the acridan were investigated. At pH 8.0 and a hydrogen peroxide concentration of 10 mM, light emission from the ECL reaction was used to determine the acridan concentration with a detection limit of 54 pmol L(-1). Results suggest that acridan esters could be used as labels in ECL immunoassays and nucleotide assays.

Sequential chemiluminescent detection of target DNAs without stripping and reprobing.

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.

CCD camera imaging for the chemiluminescent detection of enzymes using new ultrasensitive reagents.

We have designed and constructed an inexpensive imaging system based on charge-coupled device (CCD) technology and utilized it to demonstrate the sensitivity and rapid detection possible with Lumigen chemiluminescent reagents. We also report the development of two new chemiluminescent reagents, Lumi-Phos Plus and Lumigen PS. Lumi-Phos Plus is an enhanced formulation for the rapid detection of alkaline phosphatase on membranes and in solution. It provides excellent images in blotting applications with exposures of under a minute. Lumigen PS represents a new generation of peroxidase detection reagents. The wide dynamic range with excellent linearity, higher signal and lower background than other chemiluminescent reagents make Lumigen PS of unsurpassed value in enzyme-linked immunoassays and nucleic acid probe assays using HRP conjugates.

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.

Effect of photodynamic therapy on RIF-1 tumor metabolism and blood flow examined by 31P and 2H NMR spectroscopy.

Photodynamic therapy utilizes the tumor localizing drug dihematoporphyrin ether and red laser light to produce both direct tumor cell destruction via damage to mitochondrial membranes, and also indirect cell kill via destruction of the tumor vasculature. As a first step towards examining the mechanistic relationship between metabolic and vascular effects of photodynamic therapy, murine RIF-1 tumors were treated with a subcurative treatment (500 J/cm2). Tumor metabolic status was monitored using in vivo 31P NMR before, during and after the treatment. The tumor blood flow immediately before and after treatment was measured by direct intratumor injection of D2O saline and observation of the tracer signal clearance from the tumor via 2H NMR. During the photodynamic therapy treatment, significant decreases were observed for the nucleoside triphosphate concentrations, tumor pH and tumor blood flow, while inorganic phosphate concentrations increased. Animals treated with laser light alone and those not given any treatment, demonstrated no significant changes in tumor metabolic status, tumor pH or tumor blood flow. Further studies are required to determine whether tumor blood flow or metabolic status is affected first.

Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence.

A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis.

With an estimated 3-4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively, while organisms known to co-inhabit the human urogenital tract react negatively.

On the mechanism of the peroxidase-catalyzed oxygen-transfer reaction.

We reported evidence that horseradish peroxidase (HRP) and chloroperoxidase (CPO) catalyze oxygen transfer from H2O2 to thioanisoles [Kobayashi, S., Nakano, M., Goto, T., Kimura, T., & Schaap, A. P. (1986) Biochem. Biophys. Res. Commun. 135, 166-171]. In the present paper, the reaction mechanism of this oxygen transfer is discussed. The oxidation of para-substituted thioanisoles by HRP compound II showed a large negative rho value of -1.46 vs. the sigma + parameter in a Hammett plot. These results are in accord with the formation of a cation radical intermediate in the rate-determining step. Hammett treatments for HRP- and CPO-dependent S-oxygenations did not provide unequivocal proofs to judge the reaction mechanism, because of the poor correlations for sigma + and sigma p parameters. Different behavior was found in kinetics and stereoselectivity between the two enzymes. Results in the present study and recent studies strongly suggested the formation of a cation radical intermediate. The oxygen atom would transfer by reaction of compound II and the cation radical intermediate. Although involvement of the cation radical was not confirmed in the CPO system, a similar mechanism was proposed for CPO.

An evidence of the peroxidase-dependent oxygen transfer from hydrogen peroxide to sulfides.

Horseradish- and chloro-peroxidase catalyzed oxidation of sulfides have been investigated. Thioanisoles were oxygenated to the corresponding sulfoxides by such peroxidases at the expense of H2O2. Dealkylation was observed only in the chloroperoxidase-dependent oxidations of p-methoxy- and p-iso-propoxy-thioanisoles. The experiments with 18O-labeled H2O2 indicated that an oxygen atom of H2O2 is incorporated into the sulfoxides. These research lead to the conclusion that compound I or II is capable of acting as an oxygen donor as well as an electron acceptor.

Effects of hematoporphyrin (HPD) and a chemiluminescence system on the growth of transplanted tumors in C3H/HeJ mice.

Photoradiation therapy is emerging as a promising technique for combating cancer. Fundamentally, this approach consists of two steps: hematoporphyrin derivative (HPD) is used to selectively sensitize cancer cells to visible light; after an appropriate time interval, light is introduced into the tumor via a laser-fiber optic system to trigger the cytotoxic action of HPD. The present investigation was initiated to determine the therapeutic potential of HPD in combination with a chemiluminescent activator in treating mice which had been transplanted with tumors.

Effect of paraquat on cytochrome P-450-dependent lipid peroxidation in bovine adrenal cortex mitochondria.

We have investigated the effect of paraquat (methyl viologen) on lipid peroxidation in bovine adrenal cortex mitochondria. Incubation of a buffered aerobic mixture of mitochondria in the presence of Fe2+ or NADPH resulted in the formation of lipid peroxides whose accumulation could be followed at 532 nm as malondialdehyde. Fe2+ stimulates lipid peroxidation in normal mitochondria and those in which enzymes have been inactivated with heat. In contrast, NADPH has a stimulatory effect only in normal mitochondria, but not in heat-treated mitochondria. These results indicate that NADPH-dependent lipid peroxidation is an enzymatic process. Paraquat strongly inhibits this enzymatic lipid peroxidation, but has no effect on the non-enzymatic Fe2+-dependent process. The chemiluminescence that accompanies the NADPH-dependent lipid peroxidation is also markedly decreased in the presence of paraquat. Superoxide dismutase, which removes superoxide anion efficiently, does not inhibit malondialdehyde production. The mechanism of the inhibition of the lipid peroxidation by paraquat has been examined. Paraquat has no effect on NADPH-2,6-dichlorophenolindophenol reductase and on NADPH-cytochrome c reductase activities in bovine adrenal cortex mitochondria. However, paraquat strongly inhibits the NADPH-dependent reduction of cytochrome P-450. These results suggest that the inhibitory effect of paraquat on NADPH-dependent lipid peroxidation in adrenal cortex mitochondria is due to a decrease in the level of reduced cytochrome P-450 probably by diverting electrons from cytochrome P-450. Cytochrome c, which can compete with P-450 for available electrons from adrenodoxin, like paraquat had an inhibitory effect on NADPH-dependent lipid peroxidation. Lipid peroxidation was also strongly inhibited by steroid hydroxylase inhibitors, e.g., amphenone B, aminoglutethimide and metyrapone.

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria.

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.