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Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria

1971; Wiley; Volume: 19; Issue: 3 Linguagem: Inglês

10.1111/j.1432-1033.1971.tb01323.x

1432-1033

W. Wober, Petar Alaupovic,

Metabolomics and Mass Spectrometry Studies

The so‐called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids; the β‐hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A‐protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenol‐sensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the “simple” protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins. The simple protein from Serratia marcescens 08 was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties. The protein moieties from Serratia marcescens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence of a common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific O‐antigens seems to be located in the protein moiety close to lipid A.