Measurement of Protein Electrophoretic Mobility – Zetasizer Nano ZSP

Measurement of Protein Electrophoretic Mobility Using Zetasizer Nano ZSP 

 

 

Introduction  

 


   The Zetasizer Nano is a leading product in the fields of dynamic light scattering (DLS) and electrophoretic light scattering (ELS) for measuring hydrodynamic size and electrophoretic mobility.  

 

   Dynamic light scattering (DLS) is a widely used method for analyzing the characteristics of proteins and their formulations, enabling not only size measurement but also stability assessment of molecules.  

 

   Recently, with the increasing use of proteins as biological therapeutics, the stability of proteins in solution has gained considerable scientific and commercial attention. Understanding the stability and behavior of formulations is crucial for developing safe and marketable products, increasing the demand for tools that can analyze these characteristics.  

 

   Measured protein mobility by electrophoretic light scattering (ELS) serves as one of the indicators for formulating behavior and viscosity stability [1].
 

   Experimentally, measuring protein mobility presents two realistic challenges.  

 

(i) Handling protein solutions often implies dealing with diluted concentrations. The term DLS incorporates the meaning of addressing a low level of scattered light proportional to a small molecular size in small quantities. Most light scattering-based instruments released to date have lacked the sensitivity required for these measurements.  

 

(ii) Measuring protein mobility requires applying an electric field to the sample, which promotes aggregation and may cause physical damage to the protein [2]. Consequently, the mobility measurement results reflect molecular aggregation rather than the protein being measured. 

 


Three requirements are needed for accurate and precise measurement of protein mobility.
1. Equipment sensitive enough to measure both low electrophoretic mobility and low count rate related to protein dilutions
2. Measurement techniques that reduce the risk of aggregation
3. Smart automated measurement procedures that recognize and minimize aggregation occurrence  

   The Zetasizer Nano ZSP (ZSP, Image 1A) and the related software are specifically developed to meet these requirements. The light scattering sensitivity of the equipment makes it possible to measure and analyze small particles and low-concentration samples, like lysozyme with a low limit of concentration around 1mg/mL. Also, the phase analysis light scattering (PALS) technique used in the ZSP improves measurement performance for samples with low mobility, like proteins.  

 

   Scientists at Malvern Instruments found that a significant portion of aggregation effects during electrophoretic measurements occurs at the electrodes [3]. Using Malvern Instruments’ invented and patented diffusion barrier method (Figure 1B), the protein sample can be separated and protected from the cell electrodes [2, 4].  

 

   This means it is possible to apply voltage for a longer time to obtain more reliable data from the equipment [3]. The diffusion barrier method refers to the latest ASTM standards regarding mobility measurement of nanosized biomaterials [2]. 

 


   The Zetasizer software for the Zetasizer Nano ZSP includes a dedicated protocol for protein mobility measurement. This measurement method prevents sample aggregation by lowering the voltage and carefully controlling the temperature within the cell. 

 

   Before and after the measurement of mobility, the system accurately measures size using the same optical equipment (Figure 2). Hence, the measured particle size distribution directly represents the population from which mobility is measured. If a different optical device is used to measure size than that used to measure mobility, the intensity of scattered light may change, and any formed aggregates may not be detectable. Therefore, it is recommended to use the same optics for size measurement. By measuring size using the same optical equipment before and after mobility tests, the Zetasizer Nano ZSP identifies any potential aggregation occurrence during measurement. 

 


   Finally, accurate measurement of protein electrophoretic mobility allows for the calculation of protein charge based on the known relationship between protein mobility and charge [5].  

 

   The new calculator option in the Zetasizer software enables these calculations. This application note will explain the measurement of protein mobility using the ZSP. With the sensitivity of the ZSP, the efficient execution of the diffusion barrier method, and appropriate measurement protocols, the most accurate measurement values can be obtained.
 

Methods
 

   Human serum albumin (HSA) was prepared using two types of buffers to achieve a final concentration of approximately 2mg/mL each. The buffer conditions used are as follows. 

 

 1) A pH 7 buffer wherein the protein is originally dissolved, 2) pH 4.3 citrate buffer. To eliminate the influence of aggregates before starting the measurement, the sample is filtered through a 0.02μm filter.  

 

   The diffusion barrier method can prevent sample aggregation via contact with the cell electrodes. An appropriate amount of buffer is loaded into a disposable folded capillary cell. Using the end of a gel loading pipette, 20μL of HSA (2mg/ml) is loaded into the light path area at the bottom of the cell. After placing the cell in the equipment, size and zeta potential are measured. 

 

   The hydrodynamic size and mobility of HSA are measured using DLS and ELS, respectively, with the Zetasizer Nano ZSP. Both size and mobility are measured at the same scattering angle to confirm that the measured zeta potential belongs to the protein and not the aggregated material. 

 


Results 

 


    

   Figures 3A and 3D show the size distribution of the samples measured at pH 7 and pH 4.3, respectively, at the beginning of the experiments. As expected, only a single peak is confirmed in the size distribution. At pH 7, the size is 3.8nm, which is in good agreement with the reported size for HSA.  

 

   Interestingly, the size at pH 4.3 is 4.1nm, slightly larger than at pH 7. It is difficult to ascertain with this method if this indicates structural changes or higher levels of oligomerization or is an initial sign of some aggregation.  

 

   Mobility measurements for the samples then begin, and results are calculated. Figures 3B and 3E display frequency graphs from mobility measurements. Examining these frequency graphs is a helpful method for assessing measurement quality [6].  

 

   Since proteins are small, they diffuse very rapidly. During electrophoretic measurement, proteins are in an electrophoretic state; however, the diffusion rate is not completely overcome by electrophoresis.  

 

   Consequently, since the frequency shift of scattered light contains a significant diffusion component, it appears as a broad peak. As aggregates are larger, the frequency graph for samples containing aggregates features a much smaller diffusion component, resulting in much larger and sharper peaks. This is discussed in greater detail in application note MRK1651-02 and reference [4]. Here, the frequency graphs are broad and shallow, indicating that the measurements primarily focused on proteins rather than aggregates.
 

   The electrophoretic mobility measured for HSA at pH 7 is -0.88±0.2μmcm/Vs, while at pH 4.3, it is 0.44±0.2μmcm/Vs. These results confirm that the isoelectric point of the sample is between these two pH points. 

 


   Once the electrophoretic mobility is measured, the Zetasizer software’s new calculator can be used to calculate the total charge of the protein from the measured hydrodynamic size and electrophoretic mobility. Figure 4 shows that the computed HSA charge at pH 7 is approximately -18, while at pH 4.3, the calculated charge is approximately +10. 

   Figures 3C and 3F provide the size distribution of the samples after mobility measurements. The results indicate that only a minimal amount of aggregation occurred during the process.  In both cases, the main peak of the distribution is dominant, constituting 99% of the scattered light at pH 7 and 90% of the sample at pH 4.3.

 

   Thus, the results derived from this mobility suggest high reliability. The samples at pH 4.3 appeared to aggregate over time in the absence of an electric field, which is likely due to the low pH.  This effect also accounts for minor aggregation observed during the measurement.

 


Discussion 

 


   The overall results provide a mobility measurement similar to known values. Moreover, it is observed that the measured protein mobility is expectedly more certain at lower pH values. The repeatability and excellence of the measurements give strong confidence in the accuracy of the results. This is confirmed through broad frequency graphs and minimal aggregation in subsequent DLS measurements.
 

   The calculated protein charges were compared with research conducted by Tanford [2]. Tanford demonstrated that at pH 7, HSA has a valency of approximately -16, showing good agreement with the values presented here. Additionally, at pH 4.3, it was shown to have a valency of about +20. 

 

   This is certainly more definitive than the data presented here. Various possible reasons might have influenced this, including the buffers used and the source of HSA in the experiment, but the overall value alignment is clearly explained through reference [6]. 

 


   Using the Zetasizer Nano ZSP, software, and diffusion barrier method ensures reliable and precise measurements in actual measurement processes. This is achieved in three different ways. 

 


   1. The diffusion barrier method prevents aggregation of protein samples at the electrodes.
   2. The Zetasizer software automates and optimizes both size and zeta potential measurements of proteins to reduce user input and enhance measurement accuracy.
   3. The Zetasizer Nano ZSP allows measurements of size and zeta potential using the same optical set with superior sensitivity, providing users with confidence in measurement accuracy. 

 


   In conclusion, the mobility of HSA is successfully measured at 2mg/mL concentration in pH7 and pH4.3 buffers. The diffusion barrier method requires only a small amount of protein sample (20μL in this case), achieving these measurements with minimal sample cost. 

[1] Laue, Proteins in Serum – Colloids vs molecular views, presentation at Colorado Protein Stability Conference, 2011
[2] ASTM2865 – Standard guide for measurement of electrophoretic mobility and zeta potential of nanosized biological materials
[3] Corbett, Connah & Mattison, Electrophoresis. In press 2011
[4] Corbett, Connah & Mattison, Patent Pending
[5] Loeb, A.L, Wiersema, P.H. and Overbeek, (1961) “The Electrical Double Layer around a Spherical Colloid Particle” MIT Press, Cambridge Mass
[6] Corbett & Jack, Colloids & Surfaces A: Physiochemical and Engineering Aspects. (2010) 376 pp31-41