Fig. 2.

Isolation and mass and size analysis of freshly released MUC5B. A: freshly released intact MUC5B was isolated from saliva using a linear OptiPrep gradient (10–30%) in PBS as detailed in materials and methods. One-milliliter fractions were unloaded from the top of the centrifugation tube and subjected to carbohydrate analysis (periodic acid-Schiff). In this study we focused on the most rapidly sedimenting MUC5B form (III, fraction 12), which displays itself as compact granular form. B: dynamic light scattering data from an analysis of the fraction 12 (III, arrow) eluted from the Sephacryl 1000 (15 × 2.5) chromatography. The mucin was recovered in the void. To gain quantitative measurements of the mucin, the flow through the S1000 column was monitored by nine light scattering detectors and then through the refractive index detector. Data for a single angle at one degree (90°) are shown in the diagram. The average molecular weight and radius of gyration were calculated across the whole mucin peak and gave a value of mol wt 150 × 10⁶ and Rg 150 nm. C–E: electron microscopic analysis of different fractions from an OptiPrep density gradient isolation. The MUC5B was isolated using an OptiPrep gradient in which the most rapidly sedimenting material found at fraction 12 (III) is composed of very recently secreted and only partly expanded MUC5B mucin (C), whereas fractions 7–8 (pool II) are mainly dominated by the form that has proceeded further in the process of expansion from their granular organization (D). The linear fully extended form (E) is the major structure seen in fractions 4–5 (pool I). Scale bars, 100 nm. Images are representative of at least 4 different preparations. See Fig. 3 for more images.