Size-exclusion chromatography followed by triple detection array (SEC-TDA) can distinguish between oligomerisation, hydration and shape changes of proteins.
This was proven in research carried out by scientists from the Institut Pasteur in Paris, France using advanced GPC/SEC systems from Viscotek.
The team used a Viscotek Triple Detector Array (TDA302) system coupled to a GPCmax chromatographic system to characterise 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.
The results were published in the Journal of Biological Chemistry (Jan 2009; 284: 1781 - 1789).
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 analyse the eluting sample, including: a UV-visible spectrophotometer, a differential refractometer (RI), both seven-degree low angle light scattering (LALS) and 90-degree right angle light scattering (RALS) detectors, and a differential pressure (DP) wheatstone bridge viscometer.
Omnisec software analyses all the collected data and presents 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 for understanding protein binding and folding behaviours, which can be key towards understanding how these molecules interact with the human body.
An application note based on this work can be downloaded for free.
