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

Abstract

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.

Purification and Partial Biochemical Characterization of Glycoproteins in a Champenois Chardonnay Wine

“…The molecular weights (MW) of unknown molecules are calculated from the linear regression equation of log MW vs mobility. The presence of sugars is put in evidence by the periodic acid-Schiff method (PAS) (Riebe and Thorn, 1991).…”

Plant-derived recombinant human serum transferrin demonstrates multiple functions

“…This is most likely because of the addition of the 6xHis tag (900 Da) and the ER retention signal motif KDEL (600 Da) to the C‐terminus of plant‐derived rhTf. Furthermore, previous observations suggest that the molecular weight of native hTf following enzymatic cleavage of its two carbohydrate chains, as determined by SDS–PAGE, is smaller than the calculated theoretical molecular weight of hTf (Riebe and Thorn, 1991). However, the enhanced molecular weight of plant‐derived rhTf is unlikely because of the incomplete processing of the signal peptide sequence of plant‐derived rhTf.…”

Immunodetection of Proteins from Grapes and Yeast in a White Wine

“…The MWs of unknown molecules were calculated from the linear regression equation of log MW vs mobility. After electrophoresis, the separated proteins were either transferred at 4 °C to a nitrocellulose membrane using a Bio-Rad electroblotting apparatus (31) or stained with 1.5% CBB in 50% (v/v) methanol and destained in acetic acid/methanol/water (1:2:7) or stained by the PAS (32) to characterize the presence of sugars. Composition of the transfer buffer was 25 mM Tris, 190 mM glycine, and 20% (v/v) methanol.…”