Manjula Reddy
  • CSIR-Centre for Cellular and Molecular Biology, India
Research fields
  • Microbiology
Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli
Authors:  M. R. Sunayana and Manjula Reddy, date: 12/20/2015, view: 16687, Q&A: 1
2-Keto-3-deoxy-octonate (KDO) is an essential constituent of lipopolysaccharide (LPS) that forms the outermost leaflet of Gram-negative bacterial outer membrane. LPS is mainly composed of lipid A, O-antigen and a core oligosaccharide. Two molecules of KDO are present per one molecule of LPS. A proper level of LPS is required to maintain the outer membrane integrity and either high or low levels of LPS are toxic to the cell. Various methods are available for quantification of LPS; of these, determination of KDO is a simple and accurate method and it can be estimated either directly from crude bacterial cell lysates or from purified LPS by a simple colorimetric assay. Although this procedure can be theoretically used for any Gram-negative bacterium, we used it routinely to measure KDO from cell lysates of Escherichia coli (E. coli) K12 strains.
Method: The protocol is taken from Karkhanis et al. (1978). It is a simple, sensitive and reliable method to measure KDO. The assay is performed after complete acid hydrolysis of cell lysates or LPS to release the various components of LPS. Further, reaction with periodate, arsenite and thiobarbituric acid gives a pink to red color chromophore, which is measured at 548 nm after stabilizing with DMSO.
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