ABSTRACT
Capsular polysaccharides (SPS) are an integral component of gram-negative bacteria, and also have potential use as vaccine. In this paper, interactions of SPS isolated from Klebsiella strains K20 and K51 with cationic dyes pinacyanol chloride (PCYN) and acridine orange (AO) were studied by absorbance and fluorescence measurements. Both the polysaccharides having glucuronic acid as the potential anionic site induced strong metachromasy (blue shift ~100 nm) in the PCYN. The spectral changes were studied at different polymer/dye molar ratios (P/D = 0-40). A complete reversal of metachromasy was observed upon addition of co-solvents, suggesting the breakaway of dye molecules from the biopolymer matrix. Binding constant, changes in free energy, enthalpy and entropy of the dye polymer complex were also computed from the spectral data at different temperatures to reveal the nature of the interaction. Quenching of fluorescence of AO by the polymers and the incorporated mechanisms were also explored.
Subject(s)
Absorption/drug effects , Acridine Orange/metabolism , Carbocyanines/metabolism , Coloring Agents/metabolism , Ethanol/pharmacology , Klebsiella/chemistry , Polysaccharides, Bacterial/isolation & purification , Polysaccharides, Bacterial/metabolism , Spectrum Analysis , Temperature , ThermodynamicsABSTRACT
Interaction of Klebsiella K14 capsular polysaccharide with cationic dyes pinacyanol chloride, acridine orange and phenosafranin has been studied by spectrophotometric and spectrofluorometric techniques. The polymer containing both glucuronic acid and pyruvic acid in its repeating unit behaved as a unique polyelectrolyte. It induced blue shift of the absorption band of pinacyanol chloride indicating strong metachromasy. Stoichiometry of the polyanion and the dye cations in the polymer-dye compound (1:2) indicated that both glucuronic acid and pyruvic acid acted as potential anionic sites for interaction with the cationic dye molecules. The stoichiometry of anionic site (of polyanion): cationic site (of dye) in the polymer dye compound was calculated as 1:1. Interaction of the polymer with acridine orange and phenosafranin dyes studied by fluorescence measurements demonstrated Stern-Volmer type of quenching. Equivalent weight of the polymer was determined by spectrophotometric and spectrofluorometric titrations. From the present studies chromotropic property of the polymer was established.
Subject(s)
Acridine Orange/metabolism , Binding Sites , Carbocyanines/metabolism , Coloring Agents/metabolism , Klebsiella/chemistry , Phenazines/metabolism , Polysaccharides, Bacterial/chemistry , Spectrometry, Fluorescence , SpectrophotometryABSTRACT
The carbocyanine dye Stains-all displays spectral colour shifts or metachromasia upon binding to proteins, polysaccharides and other anionic substrates. The conformational status of the binding region of the substrate appears to govern the metachromatic features of the bound Stains-all. We have used this property to derive information about the conformational differences between the two anionic polysaccharides alginate and pectate. The stronger induction of circular dichroism in the 500 nm region of the dye by pectate is indicative of a greater extent of helical order in this polymer in solution than in the alginate or perhaps even hyaluronate chains.
Subject(s)
Alginates/chemistry , Carbocyanines/metabolism , Carbohydrate Conformation , Circular Dichroism , Coloring Agents/metabolism , Pectins/chemistry , Spectrophotometry , Staining and LabelingABSTRACT
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.