The degradation of mucins and polysaccharides by ascorbic acid and hydrogen peroxide,

Abstract

IT has been demonstrated [Robertson et al. 1940] that fresh vitreous humour of steers contains a labile substance capable of reducing the viscosity of synovial fluid mucin. The fact that the vitreous humour lost this activity upon standing, upon dialysis or upon addition of oxidizing agents, and the high vitamin C content of vitreous humour (approximately 05 mg./ml. as determined by 2:6-dichlorophenolindophenol titration), suggested that ascorbic acid might be an important factor in the breakdown of mucin. This hypothesis was supported by the results of an experiment in which 100 mg. of ascorbic acid added to 25 mg. of mucin in M/20 phosphate buffer pH 7-2 caused a degradation of mucin in 2 hr., as shown by a reduction of the relative viscosity from 40 to ca. 1. Upon addition of acetic 'acid, a flocculent precipitate was obtained instead of the characteristic cohesive, fibrous precipitate of normal mucin. That the reduction in viscosity was irreversible, due to a change in the size of the mucoprotein molecule, and not caused by altered characteristics of the solvent, was proved by redissolving the precipitate in phosphate buffer at pH-7-2. The degraded mucin did not produce a viscous solution as does the fibrous precipitate of normal mucin. Reduction in the size of the mucin molecule was further shown by the fact that the degraded mucin passed through a Seitz filter (EK 6) which held back normal mucin. The characteristics of the altered mucin indicated that ascorbic acid had split the original macromolecules. This breakdown may or may not have been due to hydrolysis. However, the absence of free amino-sugars or of an increased concentration of reducing substances, even after a prolonged reaction period, demonstrated that there was no further hydrolysis. The present investigation was undertaken to determine the role of ascorbic acid in the breakdown of mucin and to establish the degree of specificity of the reaction. Methods Ascorbic acid was determined by titration with 2:6-dichlorophenolindophenol in HPO3. H202 and traces of Cu salts, one or both of which were present in some of the ascorbic acid solutions, did not affect the determinations.