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Congo red fluorescence upon binding to macromolecules – a possible explanation for the enhanced intensity

Zemanek G., Jagusiak A., Chłopaś K., Piekarska B.

Bio-Algorithms and Med-Systems

2017

Vol. 13, no. 2

69--78

The present study attempts to explain the reason for the selective generation of an increase in intensity of Congo red (CR) fluorescence as an effect of the dye interacting with proteins and polysaccharides. This supramolecular dye, which creates ribbon-shaped micelles in aqueous solutions when excited with blue light (470 nm), presents low fluorescence with a maximum within the orange-red light range (approximately 600 nm). In the same conditions, CR-stained preparations of heat-denatured proteins, some native proteins (e.g. cell surface receptors) and cellulose show intense orange-red fluorescence when observed using a fluorescence microscope. The fluormetric measurements showed that the factors that cause the dissociation of the ribbon-shaped CR micelle – ethanol, urea, dimethyl sulfoxide (DMSO) and cholate – all contributed to a significant increase in the fluorescence intensity of the CR solutions. The fluorescence measurements of CR bound to the immunoglobulin light lambda (L λ) chain and soluble carboxymethyl cellulose (CMC) showed a fluorescence intensity which was many times higher. In the case of the denatured (65°C) immunoglobulin L λ chain, the fluorescence intensity significantly exceeded the values observed for the factors which break down the CR micelles. The dissociation of the ribbon-shaped micelles and the complexation of the monomeric CR form with polymers are two of the factors explaining the intense fluorescence of protein and polysaccharide preparations stained with CR.

Application of numerical analysis of fluorescence spectrum to identify properties of substances associating with Congo red micelle

Zemanek G., Jagusiak A., Kusior D., Piekarska B., Spólnik P., Stopa B.

Bio-Algorithms and Med-Systems

2011

Vol. 7, no. 2

17--23

The particles of Congo red (CR), a bis-azo dye, associate in aqueous solutions, forming ribbon-like micelles. Supramolecular dyes, as a result of their molecular size, have a limited capability of penetrating into the interior of the majority of native proteins and do not enter living cells surrounded by an intact phospholipid membrane. They may however, bind to proteins with structure destabilized by unfolding (during denaturation) or function-related changes (e.g. surface cell receptors). Congo red absorbs visible light with a maximum absorption at 500 nm, in aqueous solutions at neutral pH. After CR bind to the polymers (such as cellulose) or certain proteins, dye’s behaviour changes remarkably. These binding affects spectral properties of the dye: the absorption spectrum of the Congo red shifts towards longer wavelengths and receives a yellow-red fluorescence. A numerical analysis of the graphic data obtained from fluorescent microscope (division into channels corresponding to constituent colours and with the noise separated using a graphical program) allowed to understand better the causes of movement of the CR fluorescence spectrum and to identify the properties of substances associating with the CR micelle. By means of a statistical analysis of the data it was possible to observe that wavelength of emission and intensity of fluorescence is not only associated with the polarity of the environment but mainly with the strength of the association between the Congo red micelle and the matrix.