Enzymatic determination of free glucuronic acid with glucuronolactone reductase. II. Procedure for the enzymatic determination of glucuronic acid and its application to degradation studies of glycosaminoglycans.

An enzymatic method was devised for determination of free glucuronic acid by the use of glucuronolactone reductase from rat kidney. Free glucuronic acid in the range of 4 to 200 micrograms was determined quantitatively by this method even in the presence of neutral sugars and oligosaccharides of glycosaminoglycans. The interference due to 20 to 100 micrograms of aldohexose or aldopentose was less than 0.5% at the same concentration of free glucuronic acid. The activity of the enzyme towards substituted glucuronic acid was only 1% of that towards free glucuronic acid. The enzyme did not act on N-acetylhexosamines. The method was applied to a study of glycosaminoglycan degradation by lysosomal enzyme. When hyaluronic acid and chondroitin 4-sulfate were incubated for 24 h with a glycoprotein fraction obtained from rat liver lysosomes by concanavalin A agarose chromatography, free glucuronic acid was liberated (showing a sigmoid curve as a function of incubation time) and reached 21 and 10% of total glucuronic acid, respectively. Tetrasaccharide from chondroitin 4-sulfate was degraded to equimolar amounts of free glucuronic acid and sulfated trisaccharide.