Studying Protein Aggregation by Programmed Flow Field-Flow Fractionation Using Ceramic Hollow Fibers

Studying Protein Aggregation by Programmed Flow Field-Flow Fractionation Using Ceramic Hollow Fibers
Received for review March 7, 2005. Accepted May 9, 2005
Web Release Date: June 3, 2005
Ruohua Zhu, Wijbren Frankema, Yuli Huo, and Wim Th. Kok*
Anal. Chem.
ACS Publications
Copyright ? 2005 American Chemical Society
Polymer-Analysis Group, van't Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands, and Department of Chemistry, Capital National University, Beijing 100037, PR China
Ceramic hollow fibers have been used as separation channels for flow field-flow fractionation. The fibers were made of -alumina, with a -alumina layer on the inside wall acting as a semipermeable (ultrafiltration) membrane. The fibers and the separation system were tested by determining the diffusion coefficients of a series of standard proteins under various experimental conditions. Even for the smallest protein studied, a complete recovery from the fiber was obtained. A single fiber could be used for several months without problems such as leakage or fouling. The precision of the diffusion coefficient measurements was in the order of 5-10%. A good agreement with literature data was found. Programming of the cross-flow, with a time-delayed exponential decay program, was applied to extend the accessible size range for the sample components. With flow programming, the observed retention times increased linearly with the logarithm of the molar mass of proteins and aggregates, as predicted by theory. Heat-induced aggregation of -lactoglobulin (-LG) in aqueous solution was studied with the system. Upon heating, not only the extent of aggregation but also the size of the -LG aggregates was found to increase with the original concentration of -LG in solution and with the heating time. After heating in the presence of salt, very large aggregates were formed, with molar masses over 100 million. A multiangle light scattering detector was used to estimate molar masses and sizes of the protein aggregates. From the relation between the apparent diffusion coefficients and the molar masses of the aggregates, as well as from the ratio of the rms (scattering) and the hydrodyamic radii, it was concluded that the larger -LG aggregates behave as flexible chains in solution.
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