Implications of light scattering and secondary structure in protein concentration determination

Licentiate thesis, 2011

When using proteins in applications the amount of protein is a fundamental property. For enzyme immobilization there is a need to measure the protein concentration at a solid support. The system studied in this work is mesoporous particles. Today immobilized protein content is normally estimated from the residual protein concentration in the solution after immobilization. Any changes to the protein content for example through leakage have to be determined in the same indirect manner. It would be useful with a method to measure the actual immobilized protein content. By direct measurements on the solid material (here porous particles) with proper corrections for the disturbance from the material this could be achieved. The correction is determined by the character of the disturbance. This character can be identified by observations of absorption spectra. Use of full spectra instead of the prescribed single-wavelength measurements helps to avoid misinterpretations in protein concentration determination. This methodology was also adapted to studies of release of therapeutic proteins from drug formulations. Together with the protein polymers from the tablet matrix was released. The presence of these polymers gave rise to different disturbances in the concentration measurements and could be identified and in one case corrected for. In this work is also a study of how the protein secondary structure affects the absorption spectra in the Bradford assay. When Coomassie blue (the dye in the assay) binds to native protein there is an increase in the absorption at ~590 nm. Coomassie blue also binds to amyloid fibrils, fibrous protein structures mainly consisting of β-sheets. When binding to the fibrils the absorption shifts to longer wavelengths. By comparison to shifts caused by solvent polarity and viscosity properties of the amyloid binding sites are suggested.