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An Excel fitting routine for correcting protein absorption spectra for scatter
Tyson J Pilkington1, Matthew W Bignucolo1, Jack P Ainsworth1
1School of Natural Sciences, Laurentian University, Sudbury, Ontario, Canada.
Analytical Biochemistry
|August 5, 2023
Summary
Accurately measure protein concentration by correcting UV absorption spectra for light scattering. This novel method improves protein analysis and structural studies by accounting for Rayleigh and Mie scattering effects.
Area of Science:
- Biochemistry
- Spectroscopy
- Biophysical Chemistry
Background:
- Protein concentration is commonly determined using UV absorbance at 280 nm.
- Light scattering in protein solutions can interfere with accurate absorbance measurements.
- Existing methods may not fully correct for complex scattering phenomena.
Purpose of the Study:
- To develop a method for correcting protein UV absorption spectra for light scattering.
- To enable more accurate protein concentration determination.
- To facilitate spectral analysis of protein structural changes.
Main Methods:
- Developed an Excel Solver-based fitting routine.
- Applied the routine to correct full UV absorption spectra for Rayleigh and Mie scattering.
- Utilized protein samples with varying degrees of natural and induced scattering.
Main Results:
- The fitting method effectively corrected for light scattering.
- Improved accuracy in determining protein concentrations was achieved.
- The method demonstrated utility in analyzing spectral changes related to protein structure and scatter intensity.
Conclusions:
- A simple, effective method for correcting protein UV spectra for light scattering was established.
- This approach enhances the reliability of non-destructive protein quantification.
- The method is applicable to studying protein structural dynamics influenced by scattering.
