Effective determination of the size and charge status of biological macromolecules like proteins plays a crucial role in pharmaceutical, clinical and other biotechnological applications. In the present study, dynamic light scattering (DLS) and electrophoretic light scattering (ELS) are employed to characterize the size and charge of Lysozyme, a common protein. It is shown that the sensitivity and precision of these techniques are well suitable to discern the subtle effects of buffer solution conditions on the protein size and charge.
Methods and Terminology
Rooted in proteins primary structure, a protein solution represents a rather complex electrolyte system. For ex-ample, in a buffer solution, lysozyme molecules are surrounded with ionic “cloud” [3] (Debye-Hückel cloud, see schematic in Figure 1). They form the so-called electrostatic double layers (EDL) around a protein molecule (macro-ion): Stern layer (adjacent to protein surface) and an (outer) diffuse layer. The loosely defined border between them is called shear plane (red dash-line in Figure 1). The molecules (mostly counter-ions) within Stern layer are tightly associated and moving with lysozyme molecules. In the diffused layer, counter-/co-ion composition and, subsequently, the electrostatic potential Ψ(r) gradually approach electrical neutrality when moving away from the protein surface. The thickness of the EDL is usually characterized with the so-called Debye length (κ-1), a parameter mostly determined by solvent/buffer conditions. There are two important implications stemmed from this well-established EDL model. First, the protein hydrodynamic proper-ties, like size and charge measured with dynamic light scattering technique, should be interpreted as the physical values at the shear plane (see hydrodynamic radius Rh and zeta potential ζ in Figure 1). Secondly, the solvent/buffer conditions determine the dimension and composition of EDL, which in turn affect the hydrodynamic size and charge status of pro-teins. The effects is particularly pronounced in the cases of small proteins in low and medium salt concentrations, where value of κ·Rh is usually near unity. Therefore, accurate measurements of these protein hydrodynamic properties should be able to detect the changes of protein size and charge status due to the solvent/buffer condition variations.
In order to demonstrate this, dynamic light scattering and electrophoretic light scattering experiments are conducted on hen egg white lysozyme (HEWL) obtained commercially. Two dilute lysozyme solutions were prepared from lyophilized lysozyme (Sigma-Aldrich L6876, Lot # LSBG8654V), with lysozyme concentrations of 5 – 10 mg/mL. Sodium acetate (NaOAc) buffered acetic acid (AcOH) buffer solution was used as solvent. The buffer compositions were slightly varied for the two lysozyme solutions. The first sample (Sample-A) was dissolved in a buffer with 15 mM AcOH and 9.5 mM NaOAc, pH = 4.35. Another sample (Sample-B) was prepared in a buffer with 25 mM NaOAc/AcOH (molar-ratio 1:1), pH = 4.60. Lyophilized lysozyme powder was used as received without additional purification and directly dissolved in the buffer so-lutions. But, in order to ensure the electrostatic equilibrium of proteins in buffer solutions, both samples were dialyzed in their respective buffer for extended period of time before the light scattering measurements.
All light scattering experiments were performed at 25 °C on a NanoBrook Omni from Brookhaven Instruments Corpora-tion. DLS tests were conducted at detection angle of 90° (standard) and 173° (backscatter) to measure protein hydrody-namic radius, while protein charge was examined in phase analysis light scattering (PALS) mode at detection angle of 15° (forward).
