Quantitative Determination of Sialic Acid in the Monosialoganglioside, GM1, by the Thiobarbituric Acid Method

✍ Scribed by Roshan L. Mattoo; Saul Roseman

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 150 KB
• Volume: 246
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.9987

✦ Synopsis

The recommended procedure (8) for the hydrolysis of Several methods for the quantitative analysis of sialic glycoconjugates, including glycoproteins and ganglioacids in glycoconjugates require that the sialic acid glysides, involves heating of the sample in aqueous 0.1 N cosidic bond be cleaved prior to assay. The conven-HCl or preferably H 2 SO 4 (9) for 60 min at 80ЊC (hereintional (L. Svennerholm, (1958) Acta Chem. Scand. 12, after designated ''standard conditions''). Since incom-547-554) procedure for the hydrolysis of complex carplete release of NAN from many glycoproteins has been bohydrates such as gangliosides employs 0.1 N H / at observed under these conditions, some workers have 80ЊC for 60 min. Under these conditions, we find that extended the hydrolysis period to several hours (10), the monosialoganglioside (GM 1 ) yields less than 50% of even at the expense of partial destruction of NAN. In the total sialic acid. However, 90% recovery of sialic order to maximize recovery of NAN while minimizing acid was achieved by supplementing the hydrolysis its destruction, use of 2 M acetic acid instead of 0.1 N mixture with 0.2% sodium dodecylsulfate (SDS) and in-HCl/H 2 SO 4 for extended hydrolysis has also been advocreasing the temperature to 85ЊC. During hydrolysis un- cated (11, 12).

der these conditions, N-acetylneuraminic acid (NAN) is

We report here that less than 50% of the NAN in the degraded at about 7% per hour. If the analytical values monosialoganglioside, GM 1 , is liberated by the stanfor GM 1 are corrected for degradation, the recovery of dard conditions of hydrolysis. However, the problem NAN is essentially quantitative.