Protein Characterization Using Dynamic Light Scattering

Proteins are composed of complex polypeptide chains with unique 3-dimensional structures. These structures are stabilized by a combination of electrostatic and hydrophobic interactions, combined with a large degree of flexibility inside the structure of the molecule.

If protein solution conditions change, the molecular structure may be affected, along with a subsequent change in size. Thus monitoring the size of a protein molecule is one way of observing stability in the protein solution under its native conditions.

The sensitivity of light scattering, more specifically Dynamic Light Scattering (DLS), is shown to be ideal for protein characterization in different preparations as a pre-screen to finding optimum protein crystallization conditions. This non-invasive technique is fast, accurate, and simple to perform, and the sample can be recovered.

Popular biophysical protein characterization techniques - which are often used in conjunction with dynamic light scattering are:

• Spectroscopy (UV-Vis).
• Fluorescence.
• Circular dichroism (CD).
• Differential scanning calorimetry (DSC).
• Nuclear magnetic resonance (NMR).
• Analytical ultra centrifugation (AUC).
• Small angle x-ray spectroscopy.

In many ways the ultimate is considered to be x-ray protein crystallography as the exact 3-dimensional structure provides a unique insight into structure and function of a protein molecule. However, obtaining the protein crystals is often a time-consuming step before the structure can be determined.

	Presentations and application notes:
	On demand presentation on protein characterization using light scattering techniques. This presentation gives a general overview of different protein characterization applications. It starts with a brief introduction of the theory of static light scattering (SLS), dynamic light scattering (DLS), and electrophoretic light scattering (ELS). The concept of measuring the protein melting point by dynamic light scattering is introduced and demonstrated with hemoglobin. Thermal denaturation is delayed when the molecule is glycosylated. Molecular shape can be estimated, when the absolute molecular weight (MW) is known. Molecular interactions (for example antibody-antigen binding) or formation of quaternary structure (as shown on insulin dimer / hexamer) can be studied by dynamic light scattering and lead to more information about the protein solution. Static light scattering is used for the determination of the absolute molecular weight, and the second virial coefficient. Crystal screening using the second virial coefficient and with batch Dynamic Light Scattering is a popular application. More recently, the zeta potential has been used together with light scattering for isoelectric point and stability assessments.
	On demand presentation on Oligomers & Quaternary structure. Dynamic light scattering may be used to study the aggregation process. The term ‘aggregates’ can encompass oligomers, assembly or agglomeration. Typically, the molecular weight is measured using static light scattering. A recent, more rapid approach is the estimate from dynamic light scattering using a calibration technique. This method, for example, shows a clear difference between insulin in dimeric form at pH2 and a preparation in physiological conditions. The ‘active’ hexamer is significantly increased in size compared to the inactive dimer. Bovine serum albumin (BSA) is typically present as an equilibrium of dimer/monomer and Dynamic Light Scattering is used as a first estimate of the ratio of the two contributions.
	Application note on using static and dynamic light scattering for protein characterization The stability of a protein formulation is critical to its success as a pharmaceutical product. Non-invasive techniques such as light scattering are ideal to characterize proteins in a variety of solution conditions and obtain a profile of their state in solution. Protein solutions may exhibit thermal denaturation, changes in quaternary structure, aggregation and virial coefficient. Molecular weight and shape estimates complete the picture of the molecule. This note provides an overview of the information obtainable from light scattering studies.
	Application note on using dynamic light scattering to characterize monoclonal antibodies. Antibodies are our natural defense bodies against bacteria, viruses, and other blood borne foreign substances. Antibodies are proteins and as such prone to aggregation under perturbing solution conditions. Because of the sensitivity to small amounts of aggregates, light scattering is ideally suited to characterize antibody formulations. This application note presents measurements of the hydrodynamic size, zeta potential, absolute molecular weight and second virial coefficient of an antibody fragment (of approximately 20 kDa).
	Application note on Measurement of Proteins in solution at low concentration: Lysozyme monomer at 0.1 mg/mL. Measuring small, poorly scattering molecules in solution has been a challenge for dynamic light scattering systems. Lysozyme is a popular ‘standard protein’ used in many protein test assays. This enzyme is found for example in tears, and is responsible for killing bacterial cells. Lysozyme is relatively inexpensive and easily available. A preparation of hen egg lysozyme in sodium acetate buffer (at pH4.25) is measured at a concentration of only 0.1 mg/mL. The hydrodynamic size of the monomer is 1.9nm radius, in agreement with its crystallographic structure. Larger molecules increase light scattering and are detectable at even smaller concentrations.
	Application note on using dynamic light scattering to evaluate the of β-lactoglobulin. This application note provides a study of the electrostatic aggregation of β-lactoglobulin at low ionic strength and pH below the isoelectric point. Here, aggregation is slow enough to be evaluated by dynamic light scattering (DLS). At the onset of the experiment the beta lactoglobulin is primarily dimeric as seen from the hydrodynamic size, consistent with its expected molecular weight. Over time, the system then aggregates and the average size stabilizes at an aggregation/dimer equilibrium.
	Application note on Measurements of lipoproteins using dynamic light scattering. The particle size of low-density lipoproteins (LDL) is a significant indicator of cardiovascular disease. Dynamic light scattering is a fast and easy method to determine particle size in solution. Measurements show good agreement between light scattering and diameters obtained from gradient gel electrophoresis (GGE).
	Application note on The study of antibody - antigen interactions using dynamic light scattering. Dynamic light scattering may be used to study antibodies in solution. When the antibody binds to the antigen of a virus the z-average hydrodynamic size increases with time.
	Application note on Pharmaceutical drug development: Using dynamic light scattering to screen for promiscuous inhibitors. New lead compounds for drug development are typically discovered using high throughput combinatorial techniques that utilize screening databases of known compounds (libraries). Some of these small molecules have been shown to exhibit drug-like traits. Studies indicate that their concentration dependent inhibitory nature is a consequence of non-specific aggregation. This application note summarizes measurements performed on a candidate drug that exhibits inhibitory behavior at certain concentrations. The aggregation of the compound was examined with dynamic light scattering.