Hyaluronic Acid in Cartilage

Proteoglycans of cartilage when extracted by mild methods contain both aggregated and non-aggregated molecules (Sajdera ; 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.