Singapore, Jun 29, 2009: Research carried out by scientists from the Institut Pasteur in Paris, France, has shown that size exclusion chromatography followed by triple detection array (SEC-TDA) can distinguish between oligomerization, hydration and shape changes of proteins.

The work was conducted using advanced GPC/SEC systems from Viscotek, a company of Malvern Instruments. Headquartered in Malvern, UK, Malvern Instruments has subsidiary organizations in all major European markets, North America, China, Korea and Japan, a joint venture in India, a global distributor network and applications laboratories around the world.

Using a Viscotek Triple Detector Array (TDA302) system coupled to a GPCmax chromatographic system, the team characterized the structural and hydrodynamic properties of a fragment of the adenylate cyclase (CyaA) toxin, a major virulence factor of Bordetella pertussis, the causative bacteria of whooping cough. The research team demonstrated that calcium binding induces important hydrodynamic changes in the protein, gaining important insights into its biological function.

Conventional calibration of size exclusion chromatography (SEC) is based on known hydrodynamic volume of standard proteins. Such a calibration procedure suffers from the drawback of possible interactions between the protein of interest and the SEC matrix. Hence, conventional calibration can provide neither the molecular mass nor reliable hydrodynamic information.

The Viscotek TDA302 uses a series of detectors to analyze the eluting sample, including: a UV-Visible spectrophotometer, a differential refractometer (RI), a 7° Low Angle Light Scattering (LALS), a 90° Right Angle Light Scattering (RALS) detector and a differential pressure (DP) Wheatstone bridge viscometer. OmniSEC software analyzes all the collected data, presenting it in an information-rich format.

The UV-detector and the RI are used to measure protein concentration, which is required to determine both the absolute molecular mass (MM) and the intrinsic viscosity (IV). MM is calculated directly from the light scattering data and IV from the viscometer. The IV results give insight into protein hydration and shape. In this application, IV is the only hydrodynamic parameter significantly affected by calcium binding. The information that the system supplies is therefore essential towards understanding protein binding and folding behaviors which can be key towards understanding how these molecules interact with the human body.