Abstract
Hyaluronic acid was purified from the horny layer of guinea pigs and its biochemical and physical properties were studied. The horny layer, obtained by applying n-hexadecane to guinea pig skin, was digested with pronase, and glycosaminoglycans in the digest were separated from UV-absorbing material by Sephadex G-75 chromatography (sample A, 17.5mg). On DEAE-Sephadex chromatography, the fraction obtained with 0.5M NaCl was found to contain 94% of the total uronic acid. This fraction, consisting mainly of hyaluronic acid, was dialyzed and lyophilized (sample B, 12.5mg).
Sample B, consisting of 26.1% uronic acid and 27.0% glucosamine on a dry weight basis, could be digested completely with Streptomyces hyaluronidase. Sample B had a low reduced viscosity which showed almost no concentration dependence. The intrinsic viscosity of sample B was 0.83 dl/g and its molecular weight, calculated from its viscosity, was 34, 000. Sample B was eluted from Sepharose CL-6B as a broad peak between the void volume and the total column volume. The enzyme levels of hyaluronidase, β-glucuronidase, and β-N-acetylglucosaminidase in the n-hexadecane treated guinea pig skin increased to 1.7 to 2.5 fold those of controls after 6 days of the experiment. These results suggested that hyaluronic acid in the horny layer of n-hexadecane treated guinea pig skin might be degraded by hyaluronic acid degrading enzymes in the hyperkeratinized tissue.
