Wang F F, Kung C K, Goldwasser E
Endocrinology. 1985 Jun;116(6):2286-92. doi: 10.1210/endo-116-6-2286.
Erythropoietin (epo) is the glycoprotein hormone that induces normal red cell differentiation. Reaction of native or denatured epo with either [3H]iodoacetic acid or N-ethyl-2-[3H]maleimide did not result in the incorporation of any significant amount of radioactivity. Radiolabeling took place only if the protein were denatured before reduction and alkylation. When reduction was carried out in the presence of 6 M guanidine HCl, about 3.7 mol N-ethyl-2[3H]maleimide were covalently linked per mol epo. These results show that there are no free, accessible sulfhydryl groups in epo; there are two internal disulfide bonds. When epo was reduced in the presence of 6 M guanidine HCl and then reoxidized and the guanidine removed, about 85% of the biological activity was regenerated. The biological activity was lost irreversibly if the sulfhydryl groups were alkylated. Limited proteolysis of [3H]epo (labeled at sialic acid residues of the oligosaccharide chains) showed that it consists of two rather trypsin-resistant domains, each having a mol wt of about 16,000, connected by a small region of protein that is trypsin sensitive. The two large fragments contain most of the label. Biological activity and immunoreactivity are lost after limited tryptic proteolysis. Complexing epo with a neutralizing antibody protects its activity from proteolysis.
促红细胞生成素（EPO）是一种诱导正常红细胞分化的糖蛋白激素。天然或变性的EPO与[3H]碘乙酸或N - 乙基 - 2 - [3H]马来酰亚胺反应，均未导致任何显著量的放射性掺入。只有当蛋白质在还原和烷基化之前变性时，才会发生放射性标记。当在6M盐酸胍存在下进行还原时，每摩尔EPO约有3.7摩尔N - 乙基 - 2 - [3H]马来酰亚胺共价连接。这些结果表明，EPO中不存在游离的、可接近的巯基；存在两个内部二硫键。当EPO在6M盐酸胍存在下还原，然后再氧化并除去胍时，约85%的生物活性得以恢复。如果巯基被烷基化，生物活性会不可逆地丧失。对[3H]EPO（在寡糖链的唾液酸残基处标记）进行有限的胰蛋白酶水解表明，它由两个相当抗胰蛋白酶的结构域组成，每个结构域的分子量约为16,000，通过一个对胰蛋白酶敏感的小蛋白区域连接。两个大片段包含了大部分标记。有限的胰蛋白酶水解后，生物活性和免疫反应性丧失。用中和抗体使EPO形成复合物可保护其活性不被水解。
