Optimal conditions for lactoperoxidase catalyzed radioiodination of external proteins on mouse erythrocytes.
Optimal conditions were established for specific labelling of the surface proteins of mouse erythrocytes using lactoperoxidase-catalyzed radioiodination. The levels of H2O2 and I-, and cell concentrations required for restriction of haemoglobin labelling to less than 5% of the total 125I-protein, were different for radioiodination employing direct H2O2 addition or generation of H2O2 with glucose oxidase plus glucose. Preparation of mouse erythrocyte ghosts by hypotonic lysis caused loss of some minor labelled proteins present on intact cells and shifts to lower molecular weights of others. It is therefore important to solubilize labelled cells directly in electrophoresis buffer to avoid artifactual degradation of labelled proteins. The extent of labelling internal cell proteins was measured by a procedure suitable for the comparison of a large number of samples: solubilized radioiodinated erythrocytes were electrophoresed on 14% acrylamide gels and the radioactivity determined in the haemoglobin band which migrates separately from other proteins. The major labelled protein on the mouse erythrocytes had an apparent molecular weight of 92,000, and may be analogous to Band 3 of the human erythrocyte.
D000818:Animals; D001798:Blood Proteins / Q000032:analysis*; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis; D006861:Hydrogen Peroxide / Q000378:metabolism; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D007784:Lactoperoxidase / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism*
D001798:Blood Proteins; D006454:Hemoglobins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D007784:Lactoperoxidase; D010544:Peroxidases; D005947:Glucose