Generation of carbohydrate-deficient transferrin by enzymatic deglycosylation of human transferrin.
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-beta-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 micrograms of enzyme. This proved to be an efficient method for generating CDT.
Duan
C
C
Roche Diagnostic Systems, Somerville, NJ 08876, USA. Larryduan@compuserve.com
Rosen
S
S
Towt
J
J
Rouse
S
S
Subuhi
H
H
Salamone
S J
SJ
D000437:Alcoholism / Q000097:blood / Q000175:diagnosis; D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D002240:Carbohydrate Sequence; D002847:Chromatography, Agarose; D004800:Enzymes, Immobilized; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D014168:Transferrin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D015415:Biomarkers; D004800:Enzymes, Immobilized; D014168:Transferrin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase