Is Congo red an amyloid-specific dye?
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 alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-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.
Khurana
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R
Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, California 95064, USA.
Uversky
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VN
Nielsen
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L
Fink
A L
AL
D000682:Amyloid / Q000378:metabolism*; D001704:Biopolymers; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D004396:Coloring Agents / Q000378:metabolism*; D003208:Concanavalin A / Q000737:chemistry / Q000378:metabolism; D003224:Congo Red / Q000378:metabolism*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary
D000682:Amyloid; D001704:Biopolymers; D004396:Coloring Agents; D003208:Concanavalin A; D003224:Congo Red; D002950:Citrate (si)-Synthase; D009113:Muramidase; D011133:Polysaccharide-Lyases; C023911:pectate lyase