“Free” sialic acid in ovine submaxillary gland

Abstract

The validity of the thiobarbituric acid assay of Warren for N-acetylneuraminic acid (NANA) was examined. This method has been stated to determine both “free” of total sialic acid. About 25% of the total sialic acid present in ovine submaxillary gland homogenates is determined as “free” NANA ( i.e. gave the thiobarbituric acid reaction directly without preliminary acid hydrolysis). Only 10–15% of the so-called “free”, i.e. direct-reacting, NANA was dialyzable. If the original homogenate was subjected to the periodate oxidation step of the Warren procedure, the mixture dialyzed and the thiobarbituric acid assay completed on aliquots of the dialysis fluid, then the color developed was equivalent to almost all the direct-reacting NANA measurable before dialysis. Deproteinization of an homogenate by various means led to a fall in direct-reacting NANA that paralleled the drop in protein content. It was concluded that analysis for direct-reacting NANA in ovine submaxillary gland homogenates gives rise to artifactual “free” NANA, probably by oxidation and cleavage by periodate of NANA linked to glycoproteins, because of sopme peculier structural property or as a result of enztmic changes that take place in the whole homogenate.