Coomassie Blue Binding to Amyloid Fibrils and Native Proteins

Abstract

Coomassie brilliant blue G-250 (CB) is the dye used in the common Bradford assay for protein concentration determination. In this work we investigated the binding of CB to lysozyme and insulin in the native and amyloid fibril states by several optical spectroscopic techniques. We found that Coomassie blue binds both to the native proteins and to amyloid fibrils, but give distinctly different spectral responses. In addition, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra, and applied this understanding to the protein observations. The absorption and fluorescence spectra of CB indicated that the binding sites in the fibrils are less polar and holds 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. This was most likely due to differences in secondary structure, which was monitored by circular dichroism. Linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.