Ohtaki S, Kotani T, Nakamura Y
J Clin Endocrinol Metab. 1986 Sep;63(3):570-6. doi: 10.1210/jcem-63-3-570.
A murine monoclonal antibody (mAb) to human thyroid peroxidase (TPO) was produced and used for purification of the enzyme. This report describes studies done to characterize the mAb and the purified human TPO. The mAb bound to human TPO at a molar ratio of 1:2 or 1:1 and to human thyroid microsomes with a dissociation constant of 1.2 nM. The mAb had no effect on TPO activity to catalyze guaiacol oxidation. As immunoglobulin G, the mAb consisted of a light chain (kappa) of 26 kilodaltons (kDa) and a heavy chain (gamma 1) of 53 kDa; its pI value was 5.9. Using an mAb immunoaffinity column, 85% of TPO activity was recovered from an ammonium sulfate precipitate of a human thyroid microsomal preparation solubilized with deoxycholate. The purified TPO had a specific activity of 164 guaiacol U/mg protein and an A413 nm/A280 nm ratio of 0.26. The enzyme showed bands only at 107 and 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TPO was very stable at alkaline pH, and its ability to catalyze iodide oxidation and tyrosine iodination was increased to the same extent as its ability to catalyze guaiacol oxidation. For comparison with a conventional method, the immunoaffinity-purified TPO was trypsinized and rechromatographed on the immunoaffinity column. The trypsinized enzyme had a specific activity of 336 U/mg and an A413 nm/A280nm ratio of 0.65. The absorption spectrum of the enzyme suggested that the prosthetic group of human TPO is protoheme IX. These results indicate the value of the immunoaffinity procedure for human TPO purification. The major advantages of this purification procedure are not only high yield and speed, but also recovery of intact enzyme.
制备了一种针对人甲状腺过氧化物酶（TPO）的鼠单克隆抗体（mAb），并用于该酶的纯化。本报告描述了为表征该单克隆抗体和纯化的人TPO所做的研究。该单克隆抗体与人TPO以1:2或1:1的摩尔比结合，与人类甲状腺微粒体结合的解离常数为1.2 nM。该单克隆抗体对TPO催化愈创木酚氧化的活性没有影响。作为免疫球蛋白G，该单克隆抗体由一条26千道尔顿（kDa） 的轻链（κ）和一条53 kDa的重链（γ1）组成；其pI值为5.9。使用单克隆抗体免疫亲和柱，从用脱氧胆酸盐溶解的人甲状腺微粒体制剂的硫酸铵沉淀中回收了85%的TPO活性。纯化的TPO的比活性为164愈创木酚单位/毫克蛋白，A413 nm/A280 nm比值为0.26。该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上仅在107 kDa和100 kDa处出现条带。纯化的TPO在碱性pH下非常稳定，其催化碘化物氧化和酪氨酸碘化的能力与其催化愈创木酚氧化的能力以相同程度增加。为了与传统方法进行比较，将免疫亲和纯化的TPO用胰蛋白酶处理并在免疫亲和柱上重新色谱分离。经胰蛋白酶处理的酶的比活性为336 U/毫克，A413 nm/A280nm比值为0.65。该酶的吸收光谱表明人TPO的辅基是原卟啉IX。这些结果表明免疫亲和法在人TPO纯化中的价值。这种纯化方法的主要优点不仅是高产率和速度，而且还能回收完整的酶。
