Spectrophotometric Characteristics and Assay of Biological Stains III. The Xanthenes

In this continuation paper of the work on the chemical and spectrophotometic characteristics of commercial stains, data on the xanthene dyes are presented. In the xanthene group of dyes, it has been found possible to assay pyronin B, eosins B and Y and ethyl eosin by spectrophotometric means. Phloxine B, rose Bengal, and erythrosin B are assayed by die color acid precipitation method. Typical absorption curves are given for these dyes as well as representative spectral and assay data.     

Self-oligomerization and protein aggregation of alpha-synuclein in the presence of Coomassie Brilliant Blue.

alpha-Synuclein has been implicated in various neurodegenerative disorders, including Parkinson's and Alzheimer's diseases, by its participation in abnormal protein depositions. As the protein has been suggested to play a significant role in the formation of the deposits which might be responsible for neurodegeneration, there is a strong demand to screen for alpha-synuclein-interactive small molecules. In this report, Coomassie Brilliant Blue (CBB) interaction of alpha-synuclein has been investigated with respect to induction of protein self-oligomerization in the presence of the chemical coupling reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Both CBB-G and CBB-R, which differ by only two methyl groups, induced the self-oligomerization of alpha-synuclein in a biphasic manner with optimal dye concentrations of 250 microM and 150 microM, respectively. The protein aggregates of alpha-synuclein induced by the dyes in the absence of the coupling reagent were analysed by electron microscopy. Whereas CBB-G induced formation of protein aggregates with a worm-like structure, CBB-R induced clear fibrilization of alpha-synuclein on a background of granular structures. CBB-R interacted with alpha-synuclein approximately twice as effectively as CBB-G (dissociation constants 0.63 microM and 1.37 microM, respectively). These dye interactions were independent from the acidic C-terminus of alpha-synuclein, which was reminiscent of the Alphabeta25-35 interaction of alpha-synuclein. However, the metal-catalysed oxidative self-oligomerization of alpha-synuclein in the presence of Cu2+/H2O2, which was augmented synergistically by Alphabeta25-35, was not affected by the dyes. This indicates that the dye binding site is also distinctive from the Alphabeta25-35 interaction site on alpha-synuclein. These biochemically specific interactions between alpha-synuclein and the dyes indicate that alpha-synuclein-interactive small molecules could provide a tool with which to approach development of diagnostic, preventive, or therapeutic strategies for various alpha-synuclein-related neurodegenerative disorders.     

Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

An Evaluation of the Metachromasy of Anionic Dyes. I. Visual Observations on Tissue Sections

Thirteen dyes of the azo (benzopurpurin, Congo red, trypan blue, chromotrope 2R, orange G), indigoid (indigocarmine), triphenylmethane (acid fuchsin, aniline blue, light green, methyl blue), and xanthene (eosin B, eosin Y, erythrosin B) groups were applied under standard conditions to a variety of human, rabbit, rat, mouse and frog tissues in paraffin sections. Sections were examined for color changes which might indicate metachromatic reactions analogous to the metachromasy of cationic dyes. Disazo and xanthene dyes showed shifts in hue, with some qualification on the shifts of xanthenes. Metachromatic shifts of anionic dyes were generally of low order compared to those of cationic dyes. Nuclei, erythrocytes, inner elastic laminae of arteries, keratinous structures, and certain areas in the ground substance of connective tissue most often elicited metachromasy. It is suggested that basic proteins are responsible for the metachromatic reactions. Equally interesting areas were those staining poorly (cartilage matrix, most types of mucus), since these are sites of highly acidic substances capable of binding proteins.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Compatibility of Photoactive Dyes with Insect Biocontrol Agents

Integrated pest management (IPM) programmes often look for more specific ways to control pests. Biological control agents, such as the bacterium, Bacillus thuringiensis Berliner, and the fungus, Beauveria bassiana (Balsamo) Vuillemin, can control insects with minimal disturbance to the environment because of their host specificity and short half-lives. Often these agents alone cannot prevent yield loss or are too expensive. This study looked at the in vitro combination of these agents and photoactive dyes, especially phloxine B (red dye D&C 28), a Food and Drug Administration approved dye, with the intent to provide better insect control. Photoactive dyes are being tested for the control of many pest insects. Phloxine B and related xanthene dyes, eosin y, fluorescein and rose bengal inhibited the growth of both B. thuringiensis and B. bassiana . Phloxine B was the most inhibitory and fluorescein the least inhibitory dye for both microbes. The magnitude of inhibition increased with increasing concentration of dye and light intensity. Therefore, an adverse effect on the field performance of these biological control agents in combination with xanthene dyes would be expected.     

Lipid interaction of alpha-synuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2

Alpha-synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between alpha-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of alpha-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing beta-synuclein and a C-terminally truncated alpha-synuclein (alpha-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of alpha-synuclein. In the presence of Cu2+ and H2O2, phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of alpha-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H2O2 during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and alpha-synuclein increased from Kd of 44.7 microm to 5.9 microm in the presence of the lipid. Taken together, PI not only affects alpha-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H2O2 are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration.     

Photoinactivation of acetylcholinesterase by erythrosin B and related compounds

Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein less than eosin B less than eosin Y less than erythrosin B less than rose bengal. The observed first-order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye-enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye-enzyme dissociation constants for rose bengal (20 microM) and erythrosin B (30 microM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10(-2) M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen uptake induced by electron transfer from donors to the triplet state of methylene blue and xanthene dyes in air-saturated aqueous solution.

The effects of oxygen in the photolysis of rose bengal, eosin, erythrosin and methylene blue were studied in the presence of formate and electron donors, such as ascorbic acid, aromatic amino acids or aliphatic amines, e.g. triethylamine (TEA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide ion radical into hydrogen peroxide. The quantum yield of oxygen uptake (Phi(-O2)) increases with the donor concentration. The photoinduced formation of H2O2 is initiated by quenching of the triplet state of the dye by the donor and subsequent reactions of both the dye and donor radicals with oxygen. For methylene blue and the xanthene dyes in the presence of 10 mM ascorbic acid or 0.1 M TEA Phi(-O2)=0.07-0.25. The spectral and kinetic properties of the specific dye transients, including the radicals involved and the pH and concentration dependences, are discussed.     

Fertilization in the sea urchin, Strongylocentrotus purpuratus is blocked by fluorescein dyes.

Fertilization in gametes of the sea urchin Strongylocentrotus purpuratus was reversibly inhibited by several analogs of the anionic dye fluorescein. The dyes acted very rapidly and were effective when added before or several seconds after insemination. Eggs and sperm did not appear to be irreversibly modified by incubation in seawater solutions containing tetraiodofluorescein (erythrosin B). Sperm binding to the vitelline layer was also inhibited by erythrosin B, but required concentrations greater than that necessary to block fertilization. The ability of the compounds to block fertilization was a function of the particular fluorescein derivative used and its concentration. The concentration required to inhibit fertilization in 50% of the eggs was related to dye lipid solubility. The dyes may inhibit fertilization by preventing gamete membrane fusion.     

Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.

The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generate complementary information about the order of the system, but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. We introduce a new method that allows us to detect both signals from the same donor. Unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction, the quinoidal structure of the xanthene ring is transformed into a semiquinone, and a pi-radical is formed having a characteristic EPR signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido)fluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies.     

Envelopes for Filing Microscopic Slides

The writers discuss a series of investigations as to the behavior of certain fluorescein dyes (eosin, erythrosin, phloxine, and rose bengal) in staining bacteria in dried films of soil. These dyes are ordinarily purchased in the form of di-sodium salts and are indifferent staining agents for the purpose named. If there be added to the dye solution a small amount (0.001 to 0.1%) of a mineral salt of calcium, aluminium, magnesium or lead, the intensity of staining is greatly increased. The effect of such addition is to convert the dye partly into a salt of the metal added, which in nearly every instance is relatively insoluble and is in every case less soluble than the di-sodium salt. It is shown that practically identical results can be obtained if the staining be performed with a suspension of the calcium, aluminium or lead salt of one of these dyes, altho very little of the dye goes into solution.

Theories to account for the phenomenon are discussed, including in particular the solution and adsorption theories of staining. The evidence seems to favor the former, altho not entirely disproving the latter.     

Certain Factors Influencing the Staining Properties of Fluorescein Derivatives

The writers discuss a series of investigations as to the behavior of certain fluorescein dyes (eosin, erythrosin, phloxine, and rose bengal) in staining bacteria in dried films of soil. These dyes are ordinarily purchased in the form of di-sodium salts and are indifferent staining agents for the purpose named. If there be added to the dye solution a small amount (0.001 to 0.1%) of a mineral salt of calcium, aluminium, magnesium or lead, the intensity of staining is greatly increased. The effect of such addition is to convert the dye partly into a salt of the metal added, which in nearly every instance is relatively insoluble and is in every case less soluble than the di-sodium salt. It is shown that practically identical results can be obtained if the staining be performed with a suspension of the calcium, aluminium or lead salt of one of these dyes, altho very little of the dye goes into solution.

Theories to account for the phenomenon are discussed, including in particular the solution and adsorption theories of staining. The evidence seems to favor the former, altho not entirely disproving the latter.     

The Fluorane Derivatives: Methods for the Standardization of Biological Stains: Part II

In this paper the methods are given which are used in determining whether to approve the sale of certain dyes of the fluorane group as certified biological stains. The methods have been worked out by the Commission on Standardization of Biological Stains in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Dept. of Agriculture. The dyes for which the methods are given in the present paper are: Fluorescein, eosin yellowish, ethyl eosin, eosin bluish, erythrosin, phloxine B, and rose bengal. For each of these dyes methods are given under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of rhodamine B and kiton red S to cucurbit[7]uril: density functional investigations

The binding of the laser dyes rhodamine B (RhB) and sulforhodamine B (kiton red S or KRS) to a cucurbit[7]uril (CB[7]) host has been investigated using density functional theory. Both guests (RhB and KRS) contain two N,N-diethylamino groups on a xanthene core. The lowest-energy structure of these host-guest complexes has one of the N,N-diethylamino groups encapsulated within the host cavity, that engenders C-H···O interactions with portals, while the remaining noninteracting diethylamino group resides outside the cavity. The (1)H NMR chemical shifts derived using the gauge-independent atomic orbital method are consistent with those observed in experiments.     

Biological decolorization of xanthene dyes by anaerobic granular biomass

Biodegradation of a xanthene dyes was investigated for the first time using anaerobic granular sludge. On a first screening, biomass was able to decolorize, at different extents, six azo dye solutions: acid orange 7, direct black 19, direct blue 71, mordant yellow 10, reactive red 2 and reactive red 120 and two xanthene dyes--Erythrosine B and Eosin Y. Biomass concentration, type of electron donor, induction of biomass with dye and mediation with activated carbon (AC) were variables studied for Erythrosine B (Ery) as model dye. Maximum color removal efficiency was achieved with 4.71 g VSS L?1, while the process rates were independent of the biomass concentration above 1.89 g VSS L?1. No considerable effects were observed when different substrates were used as electron donors (VFA, glucose or lactose). Addition of Ery in the incubation period of biomass led to a fivefold increase of the decolorization rate. The rate of Ery decolorization almost duplicated in the presence of commercial AC (0.1 g L?1 AC?). Using different modified AC samples (from the treatment of AC?), a threefold higher rate was obtained with the most basic one, AC(H?), as compared with non-mediated reaction. Higher rates were obtained at pH 6.0. Chemical reduction using Na?S confirmed the recalcitrant nature of this dye. The results attest that decolorization of Ery is essentially due to enzymatic and adsorption phenomena.     

C. I. Acid Red 52: A New Stain for Cells of Granulocytic Origin

Using the xanthene dye C.I. acid red 52 (CI. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52- In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.     

C. I. acid red 52: a new stain for cells of granulocytic origin

Using the xanthene dye C.I. acid red 52 (C.I. 45100) as a single agent stain applied to coverslip preparations of blood and bone marrow, primary and secondary granules in cells of neutrophilic origin stained brilliant pink. In eosinophils, granules stained dark red. In leukemic myeloblasts that also stained with Sudan black B and demonstrated myeloperoxidase and specific esterase activity, a few bright red staining granules were visualized with acid red 52. In some leukemic promyelocytes, Auer rods stained bright red. In leukemic lymphoblasts, no red granules were seen. Of a wide variety of dyes tested so far, acid red 52 is the most sensitive stain for primary and secondary granules of granulocytes in blood and bone marrow.