Measuring the Molecular Weight and dimer % of a protein with Zetasizer Advance

Introduction

Understanding the behavior of proteins under different conditions is fundamental in several application areas such as biopharmaceuticals and health research. One parameter that can be used to understand protein behavior is absolute molecular weight (Mw). Knowing the molecular weight of a protein in monomeric form enables you to track that protein’s behavior such as dimerization and monitor the stability of your protein sample under different conditions by detecting aggregation. 

A new feature, the ‘Studies’ workspace has been added to ZS Xplorer v 3.0. Studies enables a user to conduct analysis across a series of measurements with a variance to the sample to gain a ‘bigger picture’ understanding of their sample. The first study type to be introduced is that of the ‘concentration trend’. This, as the name suggests, allows users to measure their samples at a range of concentrations to elucidate the parameters Mw, B₂₂ and K_D by both static light scattering (SLS) and dynamic light scattering. See more on the theory of SLS here. 

This application note will demonstrate how using the new concentration trends feature, a user can accurately calculate the absolute Mw of molecules in solution, and how this Mw value can be useful for detecting behavioral changes such as self-assembly in a protein sample.

Methods

Two buffer solutions of 10 mM PBS buffer were prepared and in one of the buffers 750 μM dithiothreitol was also added. Bovine serum albumin (BSA) was prepared gravimetrically in two series of concentrations of 10, 20, 30, 40 and 50 mg/ml in each of the two buffers. Samples were left to mix overnight on a hula mixer. 

Once mixed, all samples were filtered with a 0.1 μm filter and 1 ml was transferred to a DTS0012 cell. In ZS XPLORER, a concentration trend was set up and ran at 25^oC for BSA (+DTT) and BSA (-DTT).

Results 

Figure 1: Static Debye plot for BSA without DTT (trend 1 --) and with DTT (trend 2 --). The molecular weight (Mw) for each sample is equivalent to 1/y-intercept of the plot. 

Table 1: MW/ B₂₂/ K_D values for trend 1 (BSA alone) and trend 2 (BSA + DTT). 

As seen in Table 1, the molecular weight value for BSA (+DTT) is 66.55 kDa which is very close to the actual molecular weight of a BSA monomer (66.463 kDa). The molecular weight for BSA (-DTT) was measured as 80.885kDa which is higher than the molecular weight of a BSA monomer.

Discussion

BSA is a widely studied protein and is known to coexist as both monomers and dimers under certain conditions. In this experiment, BSA was prepared at fairly concentrated samples of 10-50mg/ml, which have been found to encourage BSA to form dimers via a disulfide bond. DTT is a disulfide reducing agent so should theoretically prevent BSA monomers from forming dimers. 

BSA (+DTT) had a molecular weight of 66.55 kDa which closely matches the actual molecular weight of a BSA monomer (66.46 kDa). As this measured molecular weight value is an average from all particles within the suspension, this suggests that the sample of BSA (+DTT) was monomeric. For BSA (-DTT) the molecular weight was 80.885 kDa. As the molecular weight of a BSA dimer is ~133 kDa, the results suggest that without DTT the sample of BSA contained approximately 20% dimers and 80% monomers.

Conclusion

Concentration, buffer pH and temperature can all affect the degree of BSA ‘self-assembly’ which will be reflected in its measured molecular weight. Here we have shown how concentration trends with the Zetasizer Advance instruments can be used to understand what conformation BSA is in and identify the optimum conditions for a particular use. The other output values from a concentration trend (B₂₂ and K_D) are recognized indicators of sample stability and can be used in conjunction with molecular weight to further understand the protein-protein interactions occurring.

References

1. Molecular Weight by Single Angle Debeye Plot Method | Malvern Panalytical
2. Barbosa, L. R. S. et al. The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study. Biophysical Journal 98, 147 (2010)
3. Chubarov, A. et al. Reversible Dimerization of Human Serum Albumin. Molecules 26, (2020)
4. A2 and the hydrodynamic interaction parameter k_D for assessing protein formulations | Malvern Panalytical