Influence of carbohydrate moieties of human serum transferrin on the determination of its molecular mass by polyacrylamide gradient gel electrophoresis and staining with periodic acid-Schiff reagent.
The influence of carbohydrate moieties of transferrin (Tf) on the determination of its molecular mass (MM) by polyacrylamide gradient gel electrophoresis (PAGGE) was investigated. Iron-free native human serum transferrin (Tf) of 99% purity and partly or completely carbohydrate- and N-acetylneuraminic acid (NANA)-free molecule forms were analyzed. The MM differences before and after enzymatic cleavage were found not to agree with the theoretical difference. From amino acid and carbohydrate analysis the MM of Tf was determined to be 79,570 Da whereas by denaturing and nondenaturing PAGGE MM of 77,000 Da +/- 1000 Da were found. After enzymatic cleavage of the two carbohydrate chains of Tf the difference between the calculated MM and the value reported in literature increased to 7000 Da (nondenaturing PAGGE) and 9200 Da (denaturing PAGGE). Following enzymatic cleavage of the 4 NANA molecules (MM 1237 Da) we obtained the relatively largest difference between the value given in the literature and that determined by PAGGE, namely MM 3300 Da on nondenaturing and 4000 Da on denaturing PAGGE. The differences due to the removal of the other carbohydrates were negligible. In addition we tested the periodic acid-Schiff reagent to stain iron-free Tf, containing different carbohydrate residues. The shortest carbohydrate moiety necessary for Tf staining corresponds to two identical carbohydrate chains of the structure (Asn)-GlcNAc-GlcNAc-beta-Man-(alpha-Man-)-alpha-Man.
Riebe
D
D
Institut für Biochemie und Lebensmittelchemie, Universität Hamburg, Germany.
Thorn
W
W
D000596:Amino Acids / Q000032:analysis; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D010503:Periodic Acid-Schiff Reaction; D011489:Protein Denaturation; D012794:Sialic Acids / Q000378:metabolism; D014168:Transferrin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D012794:Sialic Acids; D014168:Transferrin; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid