Hyaluronic Acid in Cartilage

Abstract

Proteoglycans of cartilage when extracted by mild methods contain both aggregated and non-aggregated molecules (Sajdera & Hascall, 1969). In the presence of 4~-guanidinium chloride aggregates were dissociated and could be separated by equilibrium densitygradient centrifugation into a glycoprotein-like fraction containing little uronic acid and a proteoglycan fraction of lower protein content (Hascall & Sajdera, 1969). On mixing of both fractions in 4~-guanidinium chloride followed by dialysis to low ionic strength, proteoglycan aggregates were re-formed. When proteoglycans from pig laryngeal cartilage were fractionated in a similar way most of the proteoglycan separated from a glycoprotein-like fraction (Tsiganos et al., 1971), but when they were mixed the proteoglycans did not reaggregate (Tsiganos et a[., 1972). In contrast, the fraction from the middle of the density gradient, which contained only 5 % of the total uronic acid and 7.2 % of the protein, when mixed with the proteoglycan produced an increase in hydrodynamic size on gel chromatography, although there was no marked change in sedimentation rate in the ultracentrifuge (R. Pain, personal communication). The effect was thus similar to but not identical with the reaggregation of subunits (Hascall & Sajdera, 1969). The component responsible for the increase in hydrodynamic size of proteoglycans has now been purified and characterized. Proteoglycans were extracted from fresh pig laryngeal cartilage with 4~-guanidinium chloride in 50m~-sodium acetate buffer, pH4.5, and purified by equilibrium densitygradient centrifugation (Tsiganos et al., 1971). The proteoglycans were fractionated in a second CsCl gradient (po = 1.5) in 4~-guanidinium chloride, in an angle rotor (8 x 25ml) in an MSE65 centrifuge, at 95000g,,. for 48h at 20°C. Three fractions, obtained from each tube by freezing and cutting, corresponded to the bottom 4nd, middle lOml and top 4ml of the gradient. The distributions of total uronic acid and protein respectively were: bottom, 92.7 and 61 7;; middle, 5.0 and 7.2%; top, 2.3 and 31.8%. The ability of samples to produce an increase in hydrodynamic size of disaggregated proteoglycans was assessed as follows. Samples were added to proteoglycans in 4 ~ guanidinium chloride, dialysed against 0.5 M-sodium acetate buffer, pH 6.8, and chroniatographed in this buffer on a column (165cm x 1.1 cm) of Sepharose 2B. The uronic acid content (Bitter & Muir, 1962) of the eluate fractions was determined by an automated method (D. Heinegard, personal communication). Since little of the disaggregated proteoglycans was eluted in the void volume before the mixing, the extent of interaction was calculated from the proportion that did so after the mixing. When the bottom and middle fractions were mixed in the same relative proportions in which they occurred in the extract, the hydrodynamic size of about half the proteoglycans increased, whereas mixing of the top and bottom fractions did not produce this effect (Tsiganos et al., 1972). A sample of the middle fraction containing 0.93 mg of uronic acid was dialysed exhaustively against distilled water, conc. HCI was added to give a concentration of 20nm and the sample was applied to a column (1 6.0cm x 1.1 cm) of ECTEOLA-cellulose (Serva, Heidelberg, Germany) previously washed with 4M-HCI followed by distilled water until the eluate was pH5. The sample was washed in with 20ml of ~ O ~ M H C I and then eluted with 20ml each of 0 .5~-NacI , 2 .5~-NaCl and ~M-HCI. Acidic fractions were neutralized as soon as they cmerged from the column, and the last two fractions were dialysed against a large excess of 0.5 M-sodium acetate. Uronic acid contents of a11 fractions were determined, and their interactions with proteoglycan were tested. Fig. I shows the distribution of uronic acid of the fractions and the ability of each to interact with proteoglycans. Most of thc uronic acid (83 %)was eluted by 2 .5~-NaCl and ~ M H C I , whereas most of the capacity to interact witfi proteoglycan (73.2%) was eluted by 0.5 r~i-NaCl. This fraction contained equimolar amounts of hexuronic acid and hexosaniine, and the glucosaiiiiiie/galactosamine molar ratio was 25 : 1.