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.     

Studies on induction of metachromasy in cationic dye pinacyanol chloride by Klebsiella K7 capsular polysaccharide.

The acidic capsular polysaccharide isolated from Klebsiella K7 induced metachromasy in the cationic dye pinacyanol chloride indicating its chromotropic character. Interaction of the biopolymer with the cationic dye was studied by visible absorption spectrophotometry, and thermodynamic parameters of the interaction evaluated. The polymer induced a metachromatic blue shift in the spectrum from 600 nm to 495 nm. The spectral changes were studied during interaction of the dye with the polymer at different polymer/dye molar ratios (P/D = 0 approximately 50). Effects of co-solvents on the stability of the dye-polymer compound were studied. A complete reversal of metachromasy was observed upon addition of different alcohols and urea solution. Thermodynamic parameters obtained from the spectral data indicated chromotropic character of the polymer in interacting with the cationic dye molecules in solution.     

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.     

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)     

Klebsiella serotype-13 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase

Periodate oxidation and Smith degradation, methylation analysis including uronic acid degradation, partial hydrolysis with acid, bacteriophage degradation, and p.m.r. spectroscopy have been used to elucidate the primary structure of the Klebsiella serotype-13 capsular polysaccharide. The polymer consists of pentasaccharide repeating-units comprising a 4)-beta-D-Manp-(1 leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to chain with a 3,4-O-(1-carboxyethylidene)-beta-D-Galp-(1 leads to 4)-alpha-D-GlcAp-(1 leads to branch at position 3 of the mannose. It is shown that there is a glycanase activity associated with particles of Klebsiella bacteriophage No. 13, which catalyses hydrolysis of chain beta-D-Glcp-(1 leads to 4)-beta-D-Manp linkages in the type-13 polysaccharide. The chemical basis of some serological cross-reactions of the Klebsiella K13 antigen is discussed.     

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).     

Studies of the primary structure of the capsular polysaccharide from Klebsiella serotype K15

The capsular polysaccharide of Klebsiella serotype K15 has been investigated mainly by methylation analysis, characterisation of the oligosaccharides obtained by partial acid hydrolysis, periodate oxidation, enzymic degradation, and 1H- and 13C-n.m.r. spectroscopy, and shown to have the hexasaccharide repeating-unit 1. The glycan does not contain any pyruvic acetal or O-acetyl substituents. (formula; see text)     

Further studies of the polysaccharide of Klebsiella pneumoniae possessing strong adjuvanticity. II. Serological relationship between the adjuvant polysaccharide and O3 antigen of Klebsiella

The serological specificity of the neutral polysaccharide possessing extraordinarily strong adjuvanticity originally isolated from the culture supernatant of Klebsiella K1 strain Kasuya has been investigated. Among all of the reference strains (K1-K82) of Klebsiella obtained from the International Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, only 13 strains have been shown to produce the adjuvant polysaccharide by the passive hemagglutination inhibition test. All of these 13 strains belong to the O3 group, and the strains which belong to other O groups of which were not identifiable did not produce it. The gel precipitation test has demonstrated that the adjuvant polysaccharide is antigenically identical to O3 antigen isolated from the cells of the decapsulated mutant (strain LEN 1) of Klebsiella K1 strain Kasuya and to O9 antigen of Escherichia coli isolated from either the culture supernatant or the cells, which has already been shown to be antigenically and structurally identical to the O3 antigen of Klebsiella.     

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.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype K 49

The structure of the capsular polysaccharide from Klebsiella type K 49 was investigated by 1H- and 13C-n.m.r. spectroscopy of the original, carboxyl-reduced, and Smith-degraded polysaccharides. Methylation of the original K 49 and derivatives showed that the polysaccharide consists of a tetrasaccharide repeating-unit having D-galacturonic as a single lateral substituent. All of the sugars have the alpha-D-configuration. This conclusion is in agreement with measurements of spin-lattice relaxation-times for the anomeric proton. O-Acetyl groups are located on galacturonic acid, but do not occupy a unique position. (Formula: see text).     

The structure of capsule polysaccharide of Klebsiella ozaenae K4 containing 3-deoxynonulosonic acid

3-Deoxy-D-glycero-D-galacto-nonulosonic acid was identified as a component of the Klebsiella ozaenae K4 capsular polysaccharide. On the basis of methylation, complete and partial acid hydrolyses, Smith degradation, and NMR analysis including computer-assisted 13C NMR evaluation, the following structure of the polysaccharide has been established.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Structure of the capsular polysaccharide of Escherichia coli O9:K32(A):H19

The capsular polysaccharide of the bacterium Escherichia coli O9:K32(A):H19 was analyzed using chemical methods (hydrolysis, sequential Smith degradation, methylation analysis) together with 1H- and 13C-n.m.r. spectroscopy. 13C-N.m.r. spectroscopy and chemical analyses indicated that the K32 polysaccharide is composed of equimolar proportions of glucose, galactose, rhamnose, and glucuronic acid, and carries O-acetyl groups. 1H-N.m.r. analysis of native K32 polysaccharide revealed five resonances in the anomeric region (delta 5.52, 5.16, 5.12, 5.02, and 4.73) and the presence of an acetyl group (delta 2.18). O-Deacetylation of the polysaccharide resulted in the loss of the resonance at delta 2.18 and one of the resonances (delta 5.52) in the anomeric region. The "extra" anomeric resonance in the 1H-n.m.r. spectrum of the native K32 polymer was assigned to H-2 of rhamnose, which experiences a large downfield shift when the 2-position is O-acetylated. This was confirmed by a 2D-COSY n.m.r. experiment and studies of model compounds. The K32 capsular polysaccharide is of the "2 + 2" type, comprised of the following repeating unit: (sequence; see text) This structure is identical to that of Klebsiella K55 capsular polysaccharide.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

The use of bacteriophage-mediated depolymerisation in the structural investigation of the capsular polysaccharide from Escherichia coli serotype K36

The structure of the repeating unit of the capsular polysaccharide from Escherichia coli serotype K36 has been established from the results of spectroscopic and chemical analyses of (a) P1, the tetrasaccharide obtained on depolymerisation of the polysaccharide with a bacteriophage-borne endo-galactosidase, (b) P1-alditol, and (c) the original polysaccharide. The repeating unit, which is identical to that reported for Klebsiella K57, has the following structure. (Formula: see text).     

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.     

Structural studies on the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide of Klebsiella serotype K40 contained D-mannose, D-glucuronic acid, D-galactose, and L-rhamnose in the approximate molar ratios 1:1:1:2. The primary structure of the capsular polysaccharide has been investigated mainly by methylation analysis, periodate oxidation, characterization of oligosaccharides, base degradation reaction, and 1H and 13CNMR spectroscopy. The polysaccharide does not contain any pyruvic acetal or O-acetyl substitution. It has a pentasaccharide repeating unit of the following primary structure: alpha-D-Manp 1----4 ----4)-beta-D-GlcpA-(1----2)-alpha-L-Rhap-(1----3)-beta-D-Ga lp-(1----2)-alpha- L-Rhap-(1----.