A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria
✍ Scribed by Yashwant D. Karkhanis; Johanna Y. Zeltner; Jesse J. Jackson; Dennis J. Carlo
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 393 KB
- Volume
- 85
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 N H,SO, at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room Temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.
Ever since its discovery as an important constituent of lipopolysaccharide (LPS) (l), there has been no satisfactory standard procedure to determine 2-keto-3-deoxyoctonic acid (KDO) in LPS. The procedure usually used is that of Osborne (2), which is a modified procedure of Weissbach and Hurwitch (3). In this procedure, a material containing LPS is hydrolyzed in a small volume of 0.02 N H,SO, at 100°C for 20 min and further treated with HIO, and NaAsO,; the formylpyruvic acid thus formed in the reaction mixture is reacted with thiobarbituric acid (TBA) to give a red chromophore which has an absorption maxima at 548 nm. Several disadvantages have been observed with this procedure; the most striking is the development of turbidity in the reaction mixture at room temperature. Although this turbidity disappears on reheating the reaction mixture, this step leads to the decomposition of the chromophore making the assay less reliable.
Since sialic acid and KDO bear some structural resemblance to each other, the procedure developed by Warren (4) for sialic acid is similar to that used for KDO. In the Warren procedure, the red chromophore is extracted with cyclohexanone or acidified butanol and its absorption is 1 To whom inquiries about the paper should be addressed.