Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography.
Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography.
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通过单克隆抗体辅助色谱法纯化的人甲状腺过氧化物酶的表征。
DOI：
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发表时间：
1986
影响因子：
5.8
通讯作者：
Y. Nakamura
中科院分区：
文献类型：
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作者：
S. Ohtaki;T. Kotani;Y. Nakamura
人甲状腺过氧化物酶（TPO）的鼠单克隆抗体（mAb）的生产和用于纯化的酶。本报告描述了为表征mAb和纯化人TPO而进行的研究。mAb以1：2或1：1的摩尔比与人TPO结合，并以1.2 nM的解离常数与人甲状腺微粒体结合。该mAb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，mAb由26千道尔顿（kDa）的轻链（κ）和53 kDa的重链（γ 1）组成;其pI值为5.9。使用mAb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物中回收85%的TPO活性。纯化的TPO具有164愈创木酚U/mg蛋白的比活性和0.26的A413 nm/A280 nm比率。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示的酶带仅在107和100 kDa。纯化的TPO在碱性pH下非常稳定，并且其催化碘化物氧化和酪氨酸碘化的能力增加到与其催化愈创木酚氧化的能力相同的程度。为了与常规方法进行比较，将免疫亲和纯化的TPO胰蛋白酶化并在免疫亲和柱上再层析。胰蛋白酶化的酶具有336 U/mg的比活性和0.65的A413 nm/A280 nm比率。该酶的吸收光谱表明，人TPO的辅基是血红素IX。这些结果表明了免疫亲和方法用于人TPO纯化的价值。这种纯化方法的主要优点是不仅产率高，速度快，而且可以回收完整的酶。
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.