Hindered convection of proteins in agarose gels

Abstract

The hindered convection of macromolecules in gels was examined by measuring the sieving coefficient (Θ, the ratio of filtrate to retentate concentration) of globular proteins in agarose membranes, as a function of protein size and gel concentration. The proteins used were lactalbumin (Stokes–Einstein radius, r s = 2.1 nm), ovalbumin ( r s = 3.0 nm), and BSA ( r s = 3.6 nm), and the volume fraction of agarose ( φ) was varied from 0.04 to 0.08. Agarose membranes were prepared on polyester mesh supports and studied in a stirred ultrafiltration cell. The Darcy permeabilities of the gels were determined in addition to Θ, and separate measurements of the mass transfer coefficient in the stirred cell were made to correct Θ for the effect of concentration polarization. The values of Θ decreased with increasing r s or φ, as expected. From the measurements of Θ and estimates of the protein diffusivity and equilibrium partition coefficient, the convective hindrance factor ( K c) was calculated for each protein–gel combination. This is the ratio of the average solute velocity (in the absence of diffusion) to the superficial fluid velocity. For small r s and φ it was found that K c slightly exceeded unity, whereas K c < 1 at larger values of r s or φ. This behavior was qualitatively, but not quantitatively, consistent with predictions from existing hindered transport theories for media consisting of parallel fibers or straight pores. Given evidence from previous partitioning and diffusion data that an agarose gel is better represented as a randomly oriented array of fibers, the large quantitative discrepancies between the data and models are not entirely surprising. Thus, the present results suggest that there is a need to extend theories of hindered convection to random arrays of fibers.