Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography. |
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Authors: | H Aoshima |
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Affiliation: | Waksman Institute of Microbiology, Rutgers University, The State University of New Jersey, P.O. Box 759, Piscataway, New Jersey 08854 USA |
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Abstract: | A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[14C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [14C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus. |
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