Diffusion and partitioning of proteins in charged agarose gels.
The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.
Johnson
E M
EM
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139-4307, USA.
Berk
D A
DA
Jain
R K
RK
Deen
W M
WM
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D002847:Chromatography, Agarose; D004058:Diffusion; D004563:Electrochemistry; D005453:Fluorescence; D005583:Fourier Analysis; D005782:Gels; D007768:Lactalbumin / Q000302:isolation & purification; D009994:Osmolar Concentration; D010047:Ovalbumin / Q000302:isolation & purification; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose; D012710:Serum Albumin, Bovine / Q000302:isolation & purification
D005782:Gels; D011506:Proteins; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D012685:Sepharose; D007768:Lactalbumin