Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light‐scattering–band‐electrophoresis apparatus

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Abstract
We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose‐gradient stabilized column, with quasielastic laser light‐scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light‐scattering apparatus by a vertical translation stage. Total intensity light‐scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ20,w = (−1.05 ± 0.05) × 10−4 cm2/V sec and D20,w = (3.35 ± 0.10) × 10−8 cm2/sec for fiberless particles, and μ20,w = −(0.59 ± 0.03) × 10−4 cm2/V sec and D20,w = (2.86 ± 0.09) × 10−8 cm2/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino‐acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.