Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation

Abstract

With dimethyl sulfoxide instead of butanol in the thiobarbituric acid assay for sialic acid, a non-fading chromophore with .lambda.max = 549 nm was produced in a homogeneous solution, allowing dilution of the test mixture in case of high color yield. This test adapted well to studies on alkaline de-O-acetylation. Bovine and rat submaxillary mucins and rabbit Tamm-Horsfall urinary sialoproteins contain O-acetyl isomers of neuraminic acid that are resistant to the thiobarbituric acid assay. Alkaline de-O-acetylation converted resistant O-acetylneuraminic acid into thiobarbituric acid-reactive sialic acid, and such conversion paralleled de-O-acetylation as measured by the ferric hydroxamate method. The color increment was similar when the alkaline treatment of bovine submaxillary mucin either preceded or followed the acid hydrolysis. Only alkaline pretreatment was effective with rat submaxillary mucin. By selecting optimal conditions for alkaline de-O-acetylation, O-acetyl isomers can be accurately assessed by the thiobarbituric acid assay.