Abstract
There are two types of light scattering measurements: static light scattering (SLS) and dynamic light scattering (DLS). The SLS method is used to estimate the molecular weight (MW) of particles by measuring the time-averaged intensity of light scattered by the particles, whereas the DLS method is used to estimate the diffusion coefficient of particles by observing the time-correlation of scattered light intensity. These techniques have recently been applied to the investigation of the aggregation, denaturation and folding, and complex formation of proteins in solution. However, the accuracy of protein size measurement by light scattering is poorly understood. In the present study, we carried out the size measurements of five globular proteins by SLS and DLS at a detection angle of 90。 and compared these data to measurements made by size exclusion chromatography (SEC). The difference (%) between the MW estimated from each method and the MW calculated from the amino acid sequence (namely the calibration residual error) was regarded as an index of measurement accuracy. The averaged calibration residual errors were 5.2 and 4.7 for SEC and SLS measurements, respectively. For the DLS measurements, the extrapolation of the apparent hydrodynamic radii to a protein concentration of zero may effectively eliminate the interparticle and hydrodynamic interactions and significantly reduced the averaged calibration residual error to 4.8%. Our results suggested that the size of globular proteins can be estimated using light scattering measurements with an accuracy equivalent to that of SEC.
Highlights
To clarify the physical properties of biomolecules, it is necessary to measure their native sizes
Our results suggest that the size of globular proteins in their physiological states can be estimated by light scattering methods with an accuracy equivalent to that obtained by size exclusion chromatography (SEC) analysis
The calibration line was obtained by the least squares method, and the differences between the MWaa calculated from the amino acid sequences and the MWSEC calibrated from Kav were used as indices of measurement accuracy
Summary
To clarify the physical properties of biomolecules, it is necessary to measure their native sizes. These methods are necessarily performed under restrictive conditions of temperature, salt concentration and pH, which are not always representative of the proteins’ native environments. Biophysical techniques such as sedimentation equilibrium analytical ultracentrifugation and small angle X-ray scattering (SAXS) are used to determine the molecular weight (MW) of proteins in their native state. These methods are complex with limited availability to most researchers. The SAXS method requires operation in a controlled area as an intense X-ray beam is used, which may result in radiation damage of the samples
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