Chromotropic character of bacterial acidic polysaccharides: Part II--Induction of metachromasy in cationic dye pinacyanol chloride by Klebsiella K10 capsular polysaccharide

The acidic capsular polysaccharide isolated from Klebsiella K10 exhibited chromotropic character with respect to induction of metachromasy in the cationic dye pinacyanol chloride (1-ethyl-2-[3-(1-ethyl-2(1H)-quinolylidene)propenyl]quinolinium chloride). Klebsiella K10 polymer consists of hexasaccharide repeating units containing one residue of glucuronic acid along with other neutral sugars in each repeating unit. It induces a metachromatic blue shift in the visible absorption spectrum of the dye from 600 nm to 500 nm. The spectral changes have been studied during interaction of the dye cations with the polyanions at different polymer/dye molar ratios. The polyanion-dye compounds are formed with polymer/dye stoichiometry of 1:1, indicating formation of stacking conformation. The complete reversal of polymer-induced metachromasy has also been observed by the addition of ethanol and urea.     

Chromotropic character of bacterial acidic polysaccharides: Part III--Interaction of cationic dye pinacyanol chloride with Klebsiella K15 capsular polysaccharide

Interaction of cationic dye pinacyanol chloride with the acidic capsular polysaccharide isolated from Klebsiella serotype K15 has been investigated by spectral measurements. Klebsiella K15 polysaccharide consists of hexasaccharide repeating units containing one residue each of glucuronic acid and glucose, and four residues of galactose. Glucuronic acid acts as the potential anionic site and the biopolymer interacts with the dye cations. It induces metachromasy in the dye and a blue shift of about 100 nm is observed in the visible absorption spectrum of the dye. Spectral measurements have been carried out at different polymer/dye molar ratios. Stoichiometry of polymer and dye in the polyanion-dye compound (1:1) indicates that every potential anionic site of the polyanion is associated with the dye cation, and stacking conformation is thus suggested. Effect of different non-aqueous solvents in reversing metachromasy has also been studied. Interaction studies exhibit chromotropic character of the biopolymer.     

Absorption and fluorescence studies on interaction between cationic dyes and Klebsiella K7 capsular polysaccharide.

Interaction of cationic dyes, pinacyanol chloride, acridine orange and phenosafranin, with Klebsiella K7 capsular polysaccharide has been investigated by spectrophotometric and spectrofluorometric measurements. The acidic polysaccharide induce a metachromatic blue shift of the absorption band of pinacyanol chloride from 600 nm to 495 nm, indicating strong metachromasy. Stoichiometry of polyanion and dye cation (1:1.5) in the polymer-dye compound formed by the interaction between pinacyanol chloride dye and K7 polymer indicate that both glucuronic acid and pyruvic acid act as the potential anionic sites for interaction. Both spectrophotometric titration of pinacyanol chloride and spectrofluorometric titration of acridine orange and phenosafranin dyes by the polymer gave quite comparable equivalent weights for the polymer. Dye-polymer interaction studies indicated induction of metachromasy in the cationic dye by the anionic biopolymer, establishing its chromotropic character.     

Metachromasy: an experimental and theoretical reevaluation

Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.     

Metachromasy: An Experimental and Theoretical Reevaluation

Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.     

An Evaluation of the Metachromasia of Bromophenol Blue

The heterogeneity of bromophenol blue from different commercial sources was revealed by paper chromatography. Isopropanol:ammonia:water (20:1:2) as the solvent system gave the best separation. A variety of impurities: violet, pink, light blue and yellow coloured ones were observed. Two of the yellow fractions showed a spectral shift to red in the presence of ammonia vapour. The respone of the main dye component with the anionic chromotropes such as heparin and hyaluronate was found to be metachromatic similar to that exhibited by the dye solution and not due to a polychromatic effect. The metachromatic effect was blocked by FeCl₃ as in the case of cationic dye metachromasy. The observed metachromatic colour is not one of the colours which characterize those resulting from changes caused by pH.     

Small molecules as chromotropes in metachromasia

Summary Interactions of cationic dye methylene blue with small polyanions like inositol hexasulfate, adenosine-triphosphate, ammonium molybdate, potassium ferro- and ferricyanide have been studied spectrophotometrically and conductometrically to ascertain the chromotropic characters of these polyanions. Results show that while almost all of them bind the dye stoichiometrically, none of them except ammonium molybdate is a chromotrope in the sense in which heparin, chondroitin sulfate etc. are. Inositol hexasulfate induces an intermediate spectra, though not perfectly metachromatic as is the case with inositol hexaphosphate. It is concluded that a chromotrope need not be a macromolecule to induce metachromasia in a dye solution, but the minimum number of charges per polyanion to give it a chromotropic character will vary with the nature of the polyanion.University Research Scholar.     

Different profiles of induced cotton effects of human and bovine lactoferrin by cibacron blue F3GA binding.

1. The properties of complex formation of lactoferrin with Cibacron Blue F3GA dye have been studied by circular dichroic spectral analyses. The Cotton effects were induced by the interaction of lactoferrin with the dye and occurred in the wavelength range from 300 to 450 nm. 2. The patterns of changes in circular dichroic spectra of lactoferrin induced by the dye were different in bovine and human lactoferrin. 3. Iron ions bound to lactoferrin affected the profiles of induced Cotton effects in human lactoferrin but not in bovine lactoferrin. 4. While the dye bound co-operatively to human apo-lactoferrin, such co-operativeness was not observed in iron-saturated human lactoferrin.     

Heterogeneity and Metachromasy of Some Commercial Anionic Dyes

The multicomponent character of all commercial anionic dyes tested (monoazo, disazo, indigoid and xanthene) was demonstrated by paper chromatography. On the basis of a reaction on filter paper, certain fractionated components of the dyes: aniline blue WS, benzoazurin, Bordeaux red, Congo red, cotton blue, chromotrope 2R, indigo-carmine, methyl-blau, soluble blue, and wasserblau showed a metachromatic response with the chromotropes, protamine and hexammine cobaltic chloride. The response of these same dye components with the chromotropes neomycin, polymyxin and viomycin was much weaker, and the alkaloids strychnine, codeine and cinchonidine could not elicit any metachromatic response. The hex-amminocobalt complex was the most effective of all the chromotropes studied, including protamine, both on filter paper and in aqueous solutions. Changes in color exhibited by the unchromatographed whole dyes such as alkali blue, alkali blue 6B, azoblau, Congo rubin, Hickson purple, isamine blue, orange G and trypan blue appear to be merely polychromatic effects because comparable changes are not shown by any of their chromatographically resolved components. In a solution system, the blue dyes, benzoazurin, cotton blue, indigo-carmine, methylblau, soluble blue, and wasserblau did not show definite visual changes in hue or in spectral shifts except with the hexamminocobalt complex, which induced a remarkable change in hue of all these dyes to a blue-violet or purple shade. A spectrophotometric study of methylblau has indicated that this change in hue is associated with a 25 mp shift of absorbance maximum to a lower wave length (hypsochromic effect). The filter-paper reaction between a dye component and a chromotrope is quite reliable and convenient for ascertaining a metachromatic response, since, unlike a reaction in solution systems, it is not affected by the unbound components of a reaction mixture. It is usable because water does not play any significant role in the metachromasy of anionic dyes. No correlation has been established between metachromasy and chemical constitution of anionic dyes.     

An Evaluation of the Metachromasy of Anionic Dyes. II. Visual and Spectral Observations on Solutions

The reactions of 13 anionic dyes in solution with a basic protein (protamine), a cationic detergent, guanidine, histamine, procaine, quinine, and strychnine were examined visually and spectrophotometrically in order to distinguish metachromatic changes of the dyes. Disazo dyes (Congo red, benzopurpurin, but not trypan blue) were metachromatic; indigoid, triphenylmethane and xanthene dyes were not. The magnitude of metachromasy in this series of dyes was not great compared with cationic dyes, the shifts of absorbance maxima being only about 15 mμ against 90 mμ or more for some cationic metachromatic dyes. The most effective chromotropes were protamine and a cationic detergent. Agreement between visual observations on tissue sections, visual observations on solutions, and spectral observations on solutions was generally good.     

Kinetic studies of interaction between acridine orange and DNA

The interaction between acridine orange (AO) and deoxyribonucleic acid (DNA) was studied by the stopped-flow method. The spectral change of AO due to interaction with DNA was followed over the wavelength range 350–600 nm at various concentration ratios of DNA phosphate to dye. The spectral change observed by the stopped-flow method was found distinctly different from that, during the dead-time, leading to a conclusion that the binding of AO to the outside of DNA occurs much faster than the intercalation into base pairs of DNA. The dependence of the rate of reaction on the reactant concentration and on the salt, concentration of the solution was also studied. The results are consistent with the mechanism that the intercalation proceeds via the outside bound state.     

A Microspectrophotometric Analysis of Staining with Carmine, Orcein and Carmine-Orcein

Microspectrophotometric measurements of carmine, orcein and carmine-orcein were made in solutions, in air-dried films and in stained adult and embryonic tissues of the domestic chicken. For individual stains only minor differences were found between dried dye and stained tissue. The absorption curve for carmine in solution showed a single peak at 490 mμ but was bimodal at about 530 and 570 mμ in dry films and stained tissue. Orcein showed a single broad peak at 510 mμ in solution; in dry films and stained tissue a broadening of the absorption curve in the red wavelengths was observed. The dye mixture carmine-orcein in solution showed a single peak at 500 mμ, but in tissue the spectral absorptions closely resembled carmine. With alum-like carmine, spectral changes due to the addition of iron were not detected. The results indicate that nuclear staining with carmine-orcein is due mainly to the carmine component of the mixture. Interpretation of spectral shifts indicates that acew-carmine is a metachromatic stain while aceto-orcein is mainly an ortho-chromatic stain, although some metachromasy is evident.     

Photophysical studies of 3,3' dioctadecyloxacarbocyanine dye in model biological membranes and different solvents

The absorption and fluorescence spectra of 3,3'-dioctadecyloxacarbocyanine [DiOC18(3)], a cationic oxacarbocyanine dye have been studied in aqueous and nonaqueous media containing egg phosphatidylcholine (PC) as well as in different solvents of diverse nature. The results show the evidence of complex formation of the dye in the ground and in the excited states with PC. The excited state interaction of the dye with PC suggests the electron transfer from PC to dye and this is supported by photovoltage generation in a photoelectrochemical cell consisting of dye and PC in aqueous medium. An attempt has been made to determine the polarity of the microenvironment of the dye in PC liposome or PC reverse micelle from the spectral studies of the dye in different solvents of known polarity.     

Spectral changes induced in Cibacron Blue F3GA by salts,organic solvents,and polypeptides: Implications for Blue dye interaction with proteins

The interactions of Cibacron Blue F3GA with organic solvents, salts, oligopeptides, and polypeptides were studied by visible difference spectroscopy. The difference spectrum of the dye in an aqueous solution of NaCl (vs water) has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Such a “salt-like” spectrum is also obtained for interaction of the dye with polycations such as oligolysines, polylysine, polyarginine, and protamine. In contrast, the difference spectrum of the dye in binary aqueous solvents containing dioxan or t-butyl alcohol at moderately high concentrations, measured against water, displays a positive peak and shoulder at 655 and 610 nm, respectively, with a small negative contribution below 550 nm. This spectrum is attributed to a nonpolar interaction of the dye with organic cosolvent molecules. The spectrum of the dye in 7 M urea is changed little from that in water, indicating similar interactions of the dye with water or urea molecules. The spectral characteristics described here for the interaction of the polyaromatic polysulfonate dye with positively charged groups, polar groups, and nonpolar moieties of neutral molecules provide a basis for describing the details of the interactions of Cibacron Blue F3GA with several proteins and for characterizing the dye binding environments in the proteins.     

A quantitative study of metachromasy in synovial fluid and mucin

Spectrophotometric measurements on synovial fluid and solutions of mucin and hyaluronate in the presence of methylene blue showed that: 1. Dialyzed synovial fluid was not metachromatic. 2. Albumin and gelatin at a concentration of 1 mg. per ml. inhibited the metachromasy of strong chromotropes. 3. Reduction of the protein of synovial fluid by the use of proteolytic enzymes still did not make the synovial fluid chromotropic. 4. Mucin solutions, with a protein content equal to that of protease-treated synovial fluid, were intensely metachromatic. 5. Sulfur-free hyaluronate produced intense metachromasy. The evidence presented indicates that in its native state in synovial fluid hyaluronate is either bound or its anionic groups are not entirely free.     

Mechanism of spectral changes of a carbocyanine dye with acidic polysaccharides and oligogalacturonides

Changes in the visible spectrum of a cationic carboeyanine dye in the presence of α (1–4) linked oligomers of d-galacturonic acid have been found to be dependent on the number of uronic acid residues in the molecule. Polygalacturonic acid caused a shift in the dye spectrum that was linearly proportional to the polymer concentration. Neither mono- nor digalacturonic acid had an effect on the dye spectrum. Tri- and tetragalacturonic acid caused spectral changes which were nonlinear with respect to oligomer concentration while penta- and hexagalacturonic acid showed concentration-dependent properties similar to polygalacturonic acid.The difference spectra with polygalacturonate and other acidic polysaccharides containing one anionic site per monosaccharide residue showed two absorption maxima in the region of 550 nm and 610 nm. All of the oligomers tested (containing 3 through 6 galacturonic acid residues) yielded only a single maxima for each in the region between 650 and 670 nm. This single maxima phenomenon was also observed with acidic polysaccharides having only one anionic site for every two monosaccharide residues (hyaluronic acid and chondroitin).     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Binding of methylene blue to alginate: Effect of acid treatment on metachromasy

The spectral properties of methylene blue (MB) in solutions of Na alginate depend on the severity of prior acid treatment of the polysaccharide. The spectral properties affected are the fraction of MB in monomeric form, the relative amounts of metachromatic dye absorbing near 570 and near 595 nm, and the intensity and sign of circular dichroism (CD) activity associated with the 570 nm bands, at various ratios of polymer equivalents to dye (P/D) from 1300 to 4. Acid treatment consisted of reaction of dry, alcohol-precipitated and presumably native alginate with 0·3m HCl at room temperature for 5 min to 8 h. Acid-induced changes showed immediate (5 min) and slow (4–8 h) stages. In both stages the fractions of MB in monomeric form and in the 595 nm metachromatic form increased. CD activity was little affected by brief acid treatment (except in range P/D=165 to 55), but diminished at all P/D values on prolonged acid treatment. Minor changes were observed in the infrared spectra of alginate films. Fresh alcohol-precipitated alginate, untreated with acid, did not precipitate when dye was in excess, nor did it form gel beads in CaCl₂ solution. It is concluded that dilute acid treatment alters the stereospecific properties of native alginate, perhaps by inducing conformational changes in the constituent copolymer segments.     

Envenomation by the box-jellyfish Chironex fleckeri: how nematocysts discharge

The capsules of isolated mastigophores of C. fleckeri were impermeable to water and neutral red dye. After air drying, ca 30% discharged, most everting fully, when exposed to distilled water or sodium citrate but in a seawater medium only partial discharge was induced. The capsular contents of discharging mastigophores dyed strongly red with neutral red and showed metachromasy with toluidine blue. Electron micrographs revealed hexagonal arrays of granules ca 12 nm in diam. Evidence supports the view that a polymerization of the capsular material initiates tubule eversion and that complete eversion involves osmotic inflow of water and sustained compression of the capsular contents.     

Enhancement of endotoxicity and reactivity with carbocyanine dye by sonication of lipopolysaccharide

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445-450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1-3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the beta band at 465 nm (dye-LPS complex) to that of the alpha band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.