Studying protein aggregation by programmed flow field-flow fractionation using ceramic hollow fibers.

Ceramic hollow fibers have been used as separation channels for flow field-flow fractionation. The fibers were made of alpha-alumina, with a gamma-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 beta-lactoglobulin (beta-LG) in aqueous solution was studied with the system. Upon heating, not only the extent of aggregation but also the size of the beta-LG aggregates was found to increase with the original concentration of beta-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 beta-LG aggregates behave as flexible chains in solution.