Application of density gradient methods for the study of mucus glycoprotein and other macromolecular components of the sol and gel phases of asthmatic sputa.

Density gradient methods have been used to characterize the sol and gel phases of sputum samples from two asthmatic patients, one extrinsic (AsEXT) and the other intrinsic (AsINT). The sputum samples were separated into sol and gel phases by ultracentrifugation (160,OOOg). Sol and gel were then separately subjected to density gradient ultracentrifugation (DGU) in CsBr to isolate mucus glycoprotein from the other macromolecular components. In both AsEXT and AsINT, sol consisted only of serum components and no mucus glycoprotein was detected. The gels too contained substantial amounts of serum proteins but had, in addition, all the mucus glycoprotein and also lipids which give the gel its opacity. While the bulk of the serum components were released from the gels in the first DGU, the lipids were strongly bound to the mucus glycoprotein, the two occurring together as insoluble fractions after the first DGU. Repeated DGU of this insoluble fraction resulted in the separation of soluble mucus glycoprotein (“native”) from the lipids. In both AsEXT and AsINT a portion of the insoluble fraction was reduced with dithiothreitol and treated with iodoacetamide prior to DGU and the “reduced” mucus glycoprotein thus isolated was also characterized. In both AsEXT and AsINT, the reduced mucus glycoprotein has lower sedimentation coefficients and solution viscosities compared to the native. Buoyant densities of the reduced mucus glycoprotein are higher than those of the corresponding native suggesting that treatment with dithiothreitol removes a peptide portion. This was confirmed by amino acid analysis. Molar ratios of carbohydrates showed no significant difference between native and reduced mucus glycoprotein, but the relative distribution of amino acids did; in both cases, serine, threonine, and proline, especially threonine, increased at the expense of aspartic acid and glutamic acid indicating that a cross-linking peptide/ protein relatively richer in the acidic amino acids takes part in the formation of disulfide-bonded aggregates in the native mucus glycoprotein.