Deep blue anthocyanins from blue Dianella berries.

Three anthocyanins, all acylated delphinidin 3,7,3',5'-tetraglucosides, and a naphthalene glycoside, 2-acetyl-1,5-dihydroxy-3 methyl-8-O(xylosyl-(1-->6)-glucosyl) naphthalene, have been isolated from the berries of two Dianella species, D. nigra and D. tasmanica. The anthocyanins show exceptional blueness at in vivo pH values due to effective intramolecular copigmentation involving p-coumaroyl-glucose units (GC) at the 7, 3' and 5' of the delphinidin anthocyanidin. Evidence is presented to show that the effectiveness of the copigmentation can be ranked; 3',5' GC>7 GC>3 GC.     

Photonuclease activity of Taylor's blue.

Taylor's blue (1,9-dimethylmethylene blue, DMMB+) associates with DNA, at least in part, through intercalation as is evidenced from the red shift in the absorption maximum, diminution of the fluorescence, and induced circular dichroism in the presence of nucleic acid. Irradiation of DMMB+/covalently closed circular supercoiled phiX174 phage DNA complex at lambda > 520 nm leads to DNA nicking in a dose-dependent manner.     

Quirks of dye nomenclature. 1. Evans blue

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.     

Quirks of dye nomenclature. 3. Trypan blue

Trypan blue is colorant from the 19^th century that has an association with Africa as a chemotherapeutic agent against protozoan (Trypanosomal) infections, which cause sleeping sickness. The dye still is used for staining biopsies, living cells and organisms, and it also has been used as a colorant for textiles.     

On the evolution of blue proteins.

Amino acid sequences of 8 plastocyanins, 8 azurins, stellacyanin, two regions in human ceruloplasmin (ferroxidase)--all of which proteins are known to bind a blue (type 1) copper--and subunit II of bovine mitochondrial cytochrome c oxidase were compared by statistical methods to assess similarities and derive possible evolutionary relationships. It is suggested that all of the examined proteins are monophyletic. The two ceruloplasmin partial sequences clearly demonstrate that this protein has undergone a duplication. A calculated most parcimonious phylogenetic tree shows the divergence of the azurin and plastocyanin ancestor to be the earliest event. Stellacyanin and later the blue oxidase (ceruloplasmin) evolved from the plastocyanin branch, which the cytochrome c oxidase subunit evolved from the azurin ancestor.     

The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae)

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4′-O-glucopyranoside as the main flavonol pigment.On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H₂O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] ⁺ at 2102 m/z (C₉₃H₁₀₅O₅₅ calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.     

Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.     

A blue dive: from ‘blue fingers’ to ‘blue silver’. A comparative overview of staining methods for in-gel proteomics

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of ‘blue silver’ colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and ‘blue silver’ CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the ‘blue silver’ method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented ‘blue silver’ can evidence the complexity of the sample.     

Dichromatism of bromphenol blue, with an improvement in the mercuric bromphenol blue technic for protein.

Staining of protein in sections using the mercuric bromphenol blue technic is improved by staining with 1% HgCl2 and 0.05% bromphenol blue in 2% aqueous acetic acid for 15 min at room temperature. Rinse slides 20 min in 2 changes of 0.5% aqueous acetic acid. Blot and give 2 fast changes in absolute ethanol with agitation before transferring to xylene. Transfer slide to 0.5% n-butylamine in xylene for a few seconds until the section is blue, then, after 2 changes of xylene, mount in DPX. Spectrophotometric analysis of this blue dye at different concentrations and with or without heparin showed that the reddish hues are due to dichromatism and not metachromasia.     

Direct effects of trypan blue on thyroid secretion.

Trypan blue directly inhibited in vitro thyroid secretion (butanol soluble 125I release to the media) induced by both thyroid stimulating hormone (TSH) and dibutyryl cAMP. Intracellular colloid droplet counts were also decreased. Inhibition was directly proportional to dye concentration and could be overcome by supramaximal TSH and dibutyryl cAMP. Inhibition could be observed as early as 20 min of incubation, was not increased by preincubation, and could even be demonstrated after TSH in vivo. Trypan blue, in vivo, produced similar inhibition of thyroid secretion. Incubation of 125I-thyroglobulin with lysosomal enzymes revealed inhibition with much lower concentrations of dye. Inhibition of lysosomal enzyme(s) would not appear to explain the marked decreases in colloid droplets, and this may represent two separate effects of trypan blue on thyroid secretion.     

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

Spectral tuning in the human blue cone pigment.

The first determination of the absolute absorption maximum of the human blue cone visual pigment is presented. After expression in COS cells, reconstitution with 11-cis-retinal, and purification, the blue pigment exhibits an absolute absorption maximum of 414 nm. The pigment reacts rapidly with hydroxylamine in the dark and is capable of activating bovine rod transducin in a light-dependent manner. Products of mutations of proposed spectral tuning residues in the blue pigment do not behave as predicted when using rhodopsin mutants as a model. Mutations of amino acids in the ring portion of the chromophore binding pocket of rhodopsin serve well as a predictive model for mutations in the blue pigment, but mutations near the Schiff base do not.