title: Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils.
abstract: Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for protein concentration determination. In this study, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CB to lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the native protein and to amyloid fibrils but gives distinctly different spectral responses. The absorption and fluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different, and this was most likely due to differences in secondary structure as monitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden. nils.carlsson@chalmers.se
authors.forename: Nils
authors.initials: N
authors.lastname: Carlsson
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Kitts
authors.forename: Björn
authors.initials: B
authors.lastname: Åkerman
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012394:Rosaniline Dyes / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis / Q000379:methods; D014783:Viscosity
chemical_list: D000682:Amyloid; D007202:Indicators and Reagents; D007328:Insulin; D012394:Rosaniline Dyes; D012997:Solvents; C048139:Coomassie blue; D009113:Muramidase