Generation of carbohydrate-deficient transferrin by enzymatic deglycosylation of human transferrin |
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Authors: | Chuanming Duan Steven Rosen James Towt Susan Rouse Haleema Subuhi Salvatore J Salamone |
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Institution: | (1) Roche Diagnostic Systems, 1080 US Highway 202, 08876 Somerville, NJ |
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Abstract: | Carbohydrate-deficient transferrin (CDT) molecules are transferrin isoforms that lack one or both of the carbohydrate groups
attached to a normal human transferrin molecule. CDT has been reported to be a sensitive and specific marker for diagnosing
alcoholism. This report demonstrates the in vitro generation of CDT molecules that can potentially be used as the standard
in measuring CDT concentrations. This was achieved by deglycosylation of human transferrin with the enzyme Endo-β-N-acetylglucosaminidase F2 (Endo-F2). The enzyme was immobilized on sepharose beads, which were packed into a column. The immobilization of the enzyme not only
eliminated the Endo-F2 contamination of CDT, but also rendered the enzyme suitable for repetitive use. In this manner, it was possible to obtain
at least 200 mg of CDT over a period of more than 3 mo, without any noticeable decrease of enzyme activity, using only 3.0
μg of enzyme. This proved to be an efficient method for generating CDT. |
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Keywords: | Carbohydrate-deficient transferrin (CDT) alcoholism Endo-β -N-acetylglucosaminidase F2 deglycosylation immobilized enzyme |
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