External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.
Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor  components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.
D000017:ABO Blood-Group System; D000328:Adult; D001894:Borohydrides; D002462:Cell Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D005260:Female; D005312:Fetal Blood / Q000032:analysis; D005692:Galactose Oxidase; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D008970:Molecular Weight; D009439:Neuraminidase; D011247:Pregnancy; D012794:Sialic Acids / Q000032:analysis; D013050:Spectrometry, Fluorescence
D000017:ABO Blood-Group System; D001894:Borohydrides; D006023:Glycoproteins; D012794:Sialic Acids; D005692:Galactose Oxidase; D009439:Neuraminidase