Student Theses and Dissertations
Date of Award
1969
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Dominic Dziewiatkowski
Keywords
proteinpolysaccharide, proteoglycan subunit, glycoprotein link, cartilage matrix, chondroitin sulfate, macromolecular aggregation
Abstract
Proteinpolysaccharides are macromolecules which consist primarily of chondroitin-4-sulfate associated with non-collagenous protein. They constitute about half of the dry weight of adult bovine nasal cartilage; the remaining material is predominantly collagen. When cartilage slices are extracted with concentrated salt solutions (typically 4 M guanidinium chloride), 85% of the total proteinpolysaccharide in the tissue is easily solubilized. The collagen framework of the tissue remains intact during the extraction procedure, and the cartilage slices retain their shapes. The amount of proteinpolysaccharide which is solubilized depends, in part, upon the ability of the extraction solvent to dissociate proteinpolysaccharide aggregates which exist in the extracellular matrix of the cartilage. In a dissociative solvent, such as 4 M guanidinium chloride, the proteinpolysaccharide in a cartilage extract is disaggregated into subunits. After the extract is dialyzed against an associative solvent such as 0.5 M guanidinium chloride, up to 60% of the proteinpolysaccharide molecules reaggregate. The relative amount of aggregate to monomer which is formed depends upon the pH of the extraction solvent; it is a maximum when extraction and aggregation are carried out at pH 5.8. Procedures which employ equilibrium centrifugation in density gradients were developed to fractionate a cartilage extract. An extract was dialyzed into an associative solvent (0.5 M guanidinium chloride buffered at pH 5.8) in order to aggregate the macromolecules. Cesium chloride was added to a density of 1.69 g/ml, and a gradient was established (40,000 rpm for 44 hours at 20° C in a Spinco SW 50 rotor). The small amount of free glycoprotein and soluble collagen in the dialyzed extract was recovered at the top of the gradient; more than 95% of the proteinpolysaccharide was recovered at the bottom in a fraction referred to as proteinpolysaccharide complex, PPC. PPC contained 10% protein, 10% keratan sulfate and 80% chondroitin sulfate. The relative amount of aggregate to subunit in PPC was the same as that in the dialyzed extract. PPC was dialyzed into a dissociative solvent (4 M guanidinium chloride buffered at pH 5.8) in order to disaggregate the macromolecules. Cesium chloride was added to a density of 1.50 g/ml, and a gradient was established (40,000 rpm for 44 hours at 20° C in a Spinco SW 50 rotor). About 30% of the protein in PPC was recovered at the top of the gradient in a fraction referred to as glycoprotein link, GPL; more than 95% of the proteinpolysaccharide was recovered at the bottom in a fraction referred to as proteoglycan subunit, PGS. PGS contained 7% protein, 8% keratan sulfate and 85% chondroitin sulfate. In associative solvent conditions, PGS contained no aggregate. PGS reaggregated when it was mixed with GPL. The amount of aggregate regenerated in a mixture was greater when the relative amount of GPL to PGS was greater. The non-covalent interactions between PGS and GPL that are required for aggregation are mediated by conformations of the protein moieties within the macromolecules. Aggregation is reversed by solvent pH below 4, by high ionic strengths such as 2 M guanidinium chloride or 3 M MgCl2 and by protein denaturants such as 1% sodium dodecylsulfate or 6 M urea. Aggregation is irreversibly abolished when cystine residues within the proteins are reduced. Although sulfhydryl reducing agents and low solvent pH prevent aggregation, they are not sufficient conditions to allow GPL to be separated from PGS. This suggests that aggregation requires at least two distinct interaction sites between GPL and PGS. Viscosimetric and centrifugal studies indicate that the PGS molecules have large effective hydrodynamic volumes (28-56 ml/g in 0.5 M guanidinium chloride), and little, if any, asymmetry (axial ratios of less than 4). The average number of PGS molecules per aggregate is about 13. The PGS fraction is polydisperse; 80% of the macromolecules have molecular weights between 1.3 and 4.1 million. This polydispersity in molecular weights is due primarily to differences in the amount of chondroitin sulfate attached to the protein moieties of the PGS macromolecules.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Hascall, Vincent C. Jr., "Proteinpolysaccharides from Bovine Nasal Cartilage Interactions with Glycoproteins and the Formation of Aggregates" (1969). Student Theses and Dissertations. 567.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/567
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy