GENERATION OF CARBOHYDRATE-DEFICIENT TRANSFERRIN BY ENZYMATIC DEGLYCOSYLATION OF HUMAN TRANSFERRIN

Carbohydrate-deficient transferrin (CDT) molecules are transferrin iso forms that lack one or both of the carbohydrate groups attached to a n ormal human transferrin molecule. CDT has been reported to be a sensit ive and specific marker for diagnosing alcoholism. This report demonst rates the in vitro generation of CDT molecules that can potentially be used as the standard in measuring CDT concentrations. This was achiev ed by deglycosylation of human transferrin with the enzyme Endo-beta-N -acetylglucosaminidase F-2 (Endo-F-2). The enzyme was immobilized on s epharose beads, which were packed into a column. The immobilization of the enzyme not only eliminated the Endo-F-2 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 mor e than 3 mo, without any noticeable decrease of enzyme activity, using only 3.0 mu g of enzyme. This proved to be an efficient method for ge nerating CDT.