DynaPro™(Protein Solutions™) Titan
Since its inception in the early 1990’s, the Protein Solutions™ DynaPro™ dynamic light scattering instrument began appearing in protein crystallization and characterization labs throughout the world – the first DLS
(Dynamic Light Scattering) technology meeting the scientific community’s need to measure small volumes of proteins under very dilute solution conditions. The DynaPro quickly earned acceptance on a global basis, and has since become an indispensable tool for biomolecular sizing and characterization. The great success of the DynaPro DLS technology is attributed to the on-going evolution in performance and capabilities designed to meet customer requirements, and is reflected by the enormous number of peer-reviewed publications citing the DynaPro.
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Recognizing the valuable contributions of the DynaPro to the research community, Wyatt Technology brought the DynaPro into it’s product line in 2004, acquiring the pertinent intellectual property from Proterion Corporation. Not satisfied to rest on its laurels, Wyatt Technology’s R&D scientists immediately began improving upon the DynaPro platform, launching the new DynaPro Titan as well as the most advanced DLS technology available today, the DynaPro Plate Reader.
The new and improved DynaPro Titan reflects Wyatt Technology’s attention to detail and customer needs including a new, easy to use DYNAMICS software interface; proprietary optics for higher sensitivity, making single mode optical fiber technology obsolete; refinements to the temperature control systems for improved response time; and ability to couple the DynaPro to Wyatt’s on-line multi-angle light scattering systems for enhanced resolution.
The DynaPro Titan when operated as a “batch” instrument supports a broad range of applications important to the characterization of biomolecules and development of therapeutic products. The DynaPro can quickly and easily size proteins, vesicles, viruses, colloids, quantum dots, and many other biomolecules and nanoparticles as a function of solution conditions and temperature. These and many other applications are available for viewing in our extensive library of
Application Notes or on-line searchable Bibliography.
However the DynaPro, as with all batch dynamic light scattering technologies, reaches a fundamental limit in its ability to resolve particles that are similar in size to each other whether proteins (oligomers, complexes), liposomes, or any of the aforementioned particles. For instance, DLS can resolve particles that are sufficiently distinct in size, as shown in Figure 1 below. Here the size distribution analysis of a sample comprising a mixture of three particles of 10, 50, and 500 nm in radius successfully determines the presence of each of the particle populations.
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Figure 1: DynaPro size distribution analysis of three polystyrene particles. Data courtesy of Narayanan Srividya, Western Michigan University.
The results of DLS are quite different than those shown in Figure 1 when analyzing a mixture of two or more species that are similar in size. While the presence different species (e.g. of oligomers (dimer, trimer, etc.) in a preparation of bovine serum albumin) may be detectable by dynamic light scattering the precise particles sizes, aggregation states, conformation, or degree of oligomerization can only be analyzed by enhancing the resolution of batch DLS.
To enhance the resolution of DLS, Wyatt’s R&D scientists developed the technology and software to couple the DynaPro to Wyatt’s existing Multi-Angle Light Scattering (MALS) instruments for operation in “chromatography mode” or “flow mode”. For all new Dynapro Titan instruments (and many previous Dynapro models!), the customer may now use the DynaPro in either the traditional “batch” manner with a cuvette, but also in flow mode simply by connecting a proprietary optical fiber from the Wyatt MALS to the DynaPro (refer to the illustration below, Figure 2).
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Figure 2: Schematic of the DynaPro connected to a Wyatt MALS system by a proprietary optical fiber. The ASTRA software controls and collects data from the combined system for flow-mode analysis.
The advantages of enhanced resolution for DLS are shown in the following experiment. A mixture of BSA (RH = 3.5 nm and MW = 64 kDa) and lysozyme (RH = 1.9 nm and MW = 14.7 kDa), was prepared, with a BSA/Lysozyme mass ratio of 4.4. The results from the regularization analysis are shown below (Figure 3). The Regularization algorithm makes no assumptions regarding the number of particle distributions. Even with the algorithm set to the maximum resolution value, the algorithm cannot resolve the two particles, reporting a weighted average radius value of 3.1 nm and a percentage polydispersity greater than 15%.
Figure 3: Radius determined from the regularization analysis from a mixture of BSA and lysozyme in PBS buffer. Note that the measured radius (3.1 nm) is between the size of lysozyme (1.9) and BSA (3.5), representing the z-average value of the two proteins. Also, a % polydispersity greater than 15% indicates the presence of unresolved species.
In some cases it is possible to determine the relative amount of each species from the batch DLS data, particularly when the system comprises two distinct populations (Lunelli, L.; Bucci, E.; Baldini, G. "Electrostatic Interactions in Hemoglobin From Light Scattering Experiments", Physical Review Letters 1993, 70(4), 513-516.).
Generally speaking, however, the limits in resolution can only be overcome by coupling the DynaPro to an HPLC system, which can be accomplished by connecting it to a Wyatt miniDAWN or DAWN system. In this configuration, the column fractionates or separates the particles by size, with each partition of the sample characterized as the it elutes through the flow cell.
Figure 4 shows a view of the intensity trace of the separation of the protein mixture using a Superose chromatographic column at a flow rate of 0.5 mL/min, and includes an overlay of the measured hydrodynamic radius. The measured sizes are consistent with the known radii of BSA and lysozyme.
Thus, only by coupling DLS with an appropriate flow mode or chromatography mode technology can particles of similar size be resolved.
Figure 4: Size and intensity trace for the BSA/Lys mixture after separation by HPLC.
Data obtained from SEC-MALS or FFF-MALS, without the addition of DLS (QELS), provides insight into the aggregation states and degree of aggregation. For small particles (less than ~ 10 nm in radius) the additional DLS size data provide several advantages.
Confirmation of the measure Rh associated with the absolute molar mass is useful when running batch experiments and tracking changes in measure Rh with solution condition. Also, the measured hydrodynamic radius and the absolute molar mass provide information the conformation of the protein via the Perrin shape factor (Cantor, Charles; Schimmel, Paul ?Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function?, Pub: W.H. Freeman and Co, New York; 1980, among others).
The combination of DLS and MALS data can provide powerful insights into the protein of interest. In the example that follows a 1:2 complex formed from Interleukin 4 Trap and Interleukin 4 eluted at a later time than the Interleukin 4 Trap, which has a lower molar mass than the complex. The explanation for this interesting behavior is explained from the DLS data which determines that the complex has a smaller hydrodynamic radius than the Interleukin 4 Trap alone.
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Please also refer to application note, effects of SEC Elution time, which presents another example of the utility applying both DLS and MALS simultaneously with HPLC.
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Wyatt Technology is the world leader in providing the most advanced macromolecular and nanoparticle characterization tools. These instruments and software may be used to determine the absolute molecular weight and or size of macromolecules and nanoparticles and include: multi-angle light scattering, dynamic light scattering, high through-put dynamic light scattering, field flow fractionation, refractive index and viscometry detection.