Showing papers by "Alexander N. Glazer published in 1990"

[31] Antioxidant activities of bile pigments: Biliverdin and bilirubin

Abstract: Publisher Summary This chapter discusses the antioxidant activities of bile pigments—biliverdin and bilirubin. Biliverdin and bilirubin are potent scavengers of singlet oxygen. The methods described in the chapter apply to the measurement of the peroxyl radical scavenging activity of biliverdin and the various biological forms of bilirubin. Most of the methods are applicable to testing the antioxidant activity of other compounds. The method is based on the thermal decomposition of an azo compound under aerobic conditions whereby peroxyl radicals are produced at a known and constant rate. In the presence of phospholipids containing polyunsaturated fatty acids (LH), the peroxyl radical will abstract a hydrogen atom to give rise to a lipid radical, thus initiating the chain reaction of lipid peroxidation. Because the peroxyl radical-mediated oxidation of polyunsaturated fatty acids initially results in quantitative formation of the corresponding lipid hydroperoxides, the extent of oxidation can be followed simply by measuring the accumulation of hydroperoxides. Any compound possessing peroxyl radical scavenging activity will compete with LH for the lipid peroxyl radical, resulting in inhibition of the chain reaction and hence inhibition of formation of lipid hydroperoxides. The results show that both biliverdin and conjugated bilirubin efficiently scavenge hypochlorous acid and act synergistically with vitamin E in protecting lipid membranes from peroxidation initiated within the lipid phase, this add further credit to the notion that the two bile pigments function as important physiological antioxidants.

[14] Phycoerythrin fluorescence-based assay for reactive oxygen species

Abstract: Publisher Summary This chapter describes the phycoerythrin fluorescence-based assay for reactive oxygen species. Phycobiliproteins are photosynthetic accessory proteins in cyanobacteria (blue-green algae) and in two groups of eukaryotic algae, the red algae and the cryptomonads. Among these proteins, B- and R-phycoerythrins are the most remarkable with respect both to the stability of their quaternary structure and to their spectroscopic properties. Both these phycoerythrins occur in numerous red algae and can be readily purified by conventional procedures. B- and R-phycoerythrins have molecular weights of 240,000 and carry 34 covalently attached tetrapyrrole prosthetic groups. Both are multisubunit proteins with the structure (αβ)6γ. The complete amino acid sequences of the α and β subunits of Porphyridium cruentum B-phycoerythrin are known, and the sequences about the tetrapyrrole attachment sites in the α, β, and γ subunits of Gastroclonium coulteri R-phycoerythrin are determined in the chapter. The assay for reactive oxygen species depends on the detection of chemical damage to phycoerythrin through the decrease in its fluorescence emission. The fluorescence of phycobiliproteins is highly sensitive to the conformation and chemical integrity of the protein and of the prosthetic groups. Under appropriate conditions, in the presence of reactive oxygen species, the rate of loss of phycoerythrin fluorescence is an index of free radical damage. The effect of added compounds on the rate of this fluorescence loss is a measure of their ability to protect the protein.

A stable double-stranded DNA-ethidium homodimer complex: Application to picogram fluorescence detection of DNA in agarose gels

Abstract: The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

Phycobiliprotein-avidin and phycobiliprotein-biotin conjugates.

Abstract: Publisher Summary Phycobiliproteins purified to homogeneity are very stable and can be stored for long periods without detectable change in aggregation state or spectroscopic properties. The amino acid sequences of numerous phycobiliproteins have been determined both directly and from analyses of cloned genes. On an average, these proteins contain about seven lysines per 100 residues and can be coupled through ɛ-amino groups to a variety of small molecules or macromolecules without any alteration in spectroscopic properties or the stability of the phycobiliprotein. Phycobiliprotein conjugates make possible fluorescence-based analyses of far higher sensitivity than that achievable with other fluorophores. Biotinylated phycoerythrin (1 ml) obtained in this manner is added slowly with stirring to 1 ml of 5 mg/ml avidin in the pH 6.8 phosphate buffer. The molar ratio of tetrameric avidin to phycoerythrin is 20. The mixture of phycoerythrin–biotin–avidin, biotin–phycoerythrin, unmodified phycoerythrin, and avidin is fractionated by high-performance liquid chromatography (HPLC) on a Varian G3000SW gel filtration column eluted with 200 mM sodium phosphate (pH 6.8).