The separation and characterization of bronchial glycoproteins by density-gradient methods

Abstract

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.