Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary cult ure. Cells were cultured under conditions that promote mucous cell differen tiation. Secreted mucins were isolated as the excluded fraction from a Seph arose CL-4B gel filtration column run under strongly dissociating condition s. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the p hysical properties of the purified mucins. The weight average molecular mas s (M-w) for three different preparations ranged from 3.3x10(6) to 4.7x10(6) g/mol (corresponding to an average structure of 1-2 subunits). and the sed imentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging fr om 4.5x10(-8) to 6.4x10(-8) cm(2)/s were calculated using the Svedberg equa tion. A polydispersity index (M-z/M-w) of similar to 1.4 was obtained. Diff usivity values were also determined by image analysis of mucin granule exoc ytosis captured by videomicroscopy. The time course of hydration and dissol ution of mucin was measured and a relationship is presented which models bo th phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution, A median diffusivity value o f 8.05x10(-8) cm2/s (inter-quartile range = 1.11x10(-7) to 6.08x10(-8) cm(2 )/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98x10(-9) cm(2)/s (inter-quartile range = 1. 47x10(-8) to 3.25x10(-9) cm(2)/sec) was determined. These values were compa red with the macromolecular diffusion coefficients (D-20,D-w) obtained by a nalytical ultracentrifugation. When differences in temperature and viscosit y were taken into account, the resulting D-37,D-g was within the range of d iffusivity values for dissolution. Our findings show that the physicochemic al properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that mea surement of the diffusivity of dissolution may be a useful means to estimat e the diffusion coefficient of mucins in mucus gel at the time of exocytosi s from a secretory cell.