Is Congo Red an Amyloid-specific Dye?

Abstract

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as α (citrate synthase), α + β (lysozyme), β (concavalin A), and parallel β-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro. Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as α (citrate synthase), α + β (lysozyme), β (concavalin A), and parallel β-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro. Congo red circular dichroism 3,3′-dithiobis(sulfosuccinimidylpropionate) dithiobis(succinimidylpropionate) initials of the patient with light chain amyloidosis whose sequence information was used to generate synthetic recombinant proteins attenuated total reflectance Fourier transform infrared spectroscopy polyacrylamide gel electrophoresis small angle x-ray scattering interleukin-2 Stokes radius In the 1920s Benhold (1Benhold H. Muenchen. Med. Wochenschr. 1922; 69: 1537Google Scholar) and Divry (2Divry P. J. Neurol. Psychiatr. 1927; 27: 643Google Scholar) established that Congo red bound to amyloid in tissue sections and demonstrated its characteristic yellow-green birefringence under crossed polarizers. Since then this birefringence has been used as a diagnostic for amyloid fibrils. The birefringence assay is not a simple one, for example, the tissue sections need to be of a required thickness to show birefringence, reviewed by Elghetany and Saleem (3Elghetany M.T. Saleem A. Stain Technol. 1988; 63: 201-212Crossref PubMed Scopus (137) Google Scholar) and Westermark et al. (4Westermark G.T. Johnson K.H. Westermark P. Methods Enzymol. 1999; 309: 3-25Crossref PubMed Scopus (237) Google Scholar). Congo red binding is not specific for amyloid in the tissue sections, but the assays are performed under extreme conditions with 50–80% ethanol, high salt and alkaline pH conditions to yield binding to amyloid (5Puchtler H. Sweat F. Levine M. J. Histochem. Cytochem. 1962; 10: 355Crossref Google Scholar). Despite these extreme conditions, binding to collagen fibers and cytoskeletal pr