Howard R J, Smith P M, Mitchell G F
Aust J Exp Biol Med Sci. 1979 Aug;57(4):355-68. doi: 10.1038/icb.1979.37.
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.
利用乳过氧化物酶催化放射性碘化法，确定了对小鼠红细胞表面蛋白进行特异性标记的最佳条件。对于直接添加H2O2或用葡萄糖氧化酶加葡萄糖产生H2O2的放射性碘化法，将血红蛋白标记限制在总125I蛋白的5%以下所需的H2O2和I-水平以及细胞浓度是不同的。通过低渗裂解制备小鼠红细胞血影会导致完整细胞上存在的一些少量标记蛋白丢失，而其他一些蛋白的分子量则向更低方向偏移。因此，直接将标记细胞溶解在电泳缓冲液中很重要，以避免标记蛋白的人为降解。通过一种适用于大量样品比较的方法来测量细胞内蛋白的标记程度：将溶解的放射性碘化红细胞在14%丙烯酰胺凝胶上进行电泳，并在与其他蛋白分开迁移的血红蛋白条带中测定放射性。小鼠红细胞上的主要标记蛋白的表观分子量为92,000，可能类似于人类红细胞的带3蛋白。
