Measuring molecular interactions in solution using multi-wavelength analytical ultracentrifugation: combining spectral analysis with hydrodynamics

Abstract

In 1926, the Swedish scientist Theodor Svedberg was awarded the Nobel Prize in Chemistry for his work on a disperse system, and for studying the colloidal properties of proteins. This work was, to a large extent, made possible by his invention of a revolutionary tool, the analytical ultracentrifuge. These days, technological advances in hardware and computing have transformed the field of analytical ultracentrifugation (AUC) by enabling entirely new classes of experiments and modes of measurement unimaginable by Svedberg, making AUC once again an indispensable tool for modern biomedical research. In this article these advances and their impact on studies of interacting molecules will be discussed, with particular emphasis on a new method termed multi-wavelength analytical ultracentrifugation (MWL-AUC). Novel detectors allow us to add a second dimension to the separation of disperse and heterogeneous systems: in addition to the traditional hydrodynamic separation of colloidal mixtures, it is now possible to identify the sedimenting molecules by their spectral absorbance properties. The potential for this advance is significant for the study of a large range of systems. A further advance has occurred in data management and computational capabilities, opening doors to improved analysis methods, as well as direct networking with the instrument, facilitating data acquisition and data handling, and significant increases in data density from faster detectors with higher resolution capability.

Figure 1

Schematic of an AUC experiment. A light source illuminates a sedimenting and diffusing solution of molecules in a sector-shaped compartment while the rotor is spinning. Different optical systems collect data representing concentration distributions of the sample as a function of time and radius.

Figure 2

Mixtures of protein and DNA. When DNA (red) and protein (blue) are mixed, the spectra of each molecule combine in an additive fashion to produce a new spectrum for the mixture (green).

Figure 3

A single scan from a 3D multi-wavelength experiment. Each wavelength mapped on the y-axis represents a separate boundary typically observed in a traditional single-wavelength experiment. Each radial position, mapped on the x-axis, provides a complete wavelength scan of the species sedimenting at that radial position. At approximately 6.1 cm the meniscus of the solution column is visible. The absorbance of this surface is mapped to the z-axis.