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Vimr purpose6/22/2023 To designate acetylation of Neu5Ac, the abbreviations Neu5,7Ac 2, Neu5,8Ac 2, Neu5,9Ac 2, or Neu4,5Ac 2 identify O-acetyl groups at the indicated carbon positions, respectively. Our modified methods describe the use of the DMB labeling procedure for analyzing bacterial polysialic acids. Hara and associates first described the DMB labeling method to measure the degree of O-acetylated sialic acids in serum glycoproteins ( 10). The degree of O-acetylation is quantified by reacting the resulting alpha-keto acids with DMB, giving rise to fluorescent quinoxalinones that are separated by reverse phase chromatography. Modified capsule chains are ultimately transported to the outer surface of the outer bacterial membrane where the constituent sialic acids, either with or without prior purification, can be analyzed after enzymatic or chemical hydrolysis of the polysaccharides. The enzyme uses acetyl-coenzyme A as donor to transfer acetyl groups to Neu5Ac residues during polymerization of the nascent polysialic acid chains inside the cell. The variable O-acetylation at carbons-7 or -9 is controlled by a random translational switch and expression of a specific polysialic acid O-acetyltransferase encoded by neuO ( 4- 9). Therefore, attention must be given to how the bacteria are grown and handled during preparation for subsequent analysis by labeling with 1,2-diamino-4,5-methylenedioxy-benzene (DMB) ( Fig. These groups, but not the acetamido at carbon-5, are base labile and can be lost during analytical or preparative procedures. ![]() coli K1 isolates are variably modified with O-acetyl groups (CH 3-COO-) attached to carbon-7 or -9 of the Neu5Ac monomer ( Fig. These methods are described in his article. We have adapted a variety of preparative and release procedures that allow chemical analyses of virtually any sample regardless of its purity ( 4). ![]() ![]() Growth in highly buffered defined chemical medium maximizes the amount of capsule retained at the cell surface ( 3). The chains are attached to the surface by an acid labile phosphodiester bond such that, depending on growth conditions, the capsule is continually sloughed into the medium. The Escherichia coli capsular polysialic acid chains are homopolymers composed of about 200 sialic acid (Neu5Ac) residues connected by alpha-2,8-glycoketosidic linkages. Capsules are usually linked to the bacterial cell surface and are most often composed of one or two different carbohydrates. Capsular polysaccharides represent one class of cell surface structure that receives intensive research scrutiny due to its value as target for vaccine and new drug development ( 1, 2). The physiochemical, immunological, and host-interactive properties of bacterial cell surfaces are dominated by polysaccharides composed of one or more types of carbohydrate monomers.
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