Reichert L E, Ramsey R B
J Biol Chem. 1975 Apr 25;250(8):3034-40.
Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.
利用大鼠睾丸组织受体分析来研究在不同尿素浓度和pH条件下人促卵泡激素（hFSH）和促黄体生成素（hLH）的解离动力学。在这些竞争性蛋白质结合分析中，125I - hFSH和125I - hLH作为放射性配体，激素解离通过解离激素抑制组织受体摄取放射性配体能力的下降来跟踪。通过非线性回归分析，分析促性腺激素解离的速率数据与一级或二级积分速率方程的拟合质量。在pH 8、25℃条件下用4 M尿素处理hFSH 22小时未导致显著解离，而在8 M尿素中观察到超过90%的解离。将温度从25℃提高到37℃，hFSH在8 M尿素中的解离速率增加约4倍。当通过使用公认的FSH全动物生物测定法跟踪hFSH的解离时，获得了类似的结果，从而证实了组织受体分析用于此类解离研究的可靠性。动力学研究表明，在25℃下于pH 8的6 M尿素中孵育4小时后，hFSH未解离。相比之下，在类似条件下hLH解离了90%。hLH这种不同的失活速率使得能够制备出具有显著降低的LH活性污染水平的hFSH，这通过组织受体分析和全动物生物测定法得以确定。在酸性条件下，hFSH和hLH的解离速率存在明显差异。在pH 2（25℃）孵育约2分钟后hFSH完全解离，在pH 3孵育15分钟后超过90%解离。相比之下，在pH 2（25℃）孵育20分钟后hLH解离60%，在pH 3孵育60分钟后解离40%。在pH 4.4孵育60分钟后，两种激素均未显著解离，但在该pH孵育1至23小时期间，hFSH的解离速率略高于LH。在pH 12孵育1小时后，hFSH和hLH相对抗解离，但在该pH孵育22小时后显著解离。上述各种条件下hFSH或hLH的解离时间进程均未明显符合一级或二级动力学，表明总体解离过程代表混合级反应。似乎在hLH和hFSH的一个或两个亚基解离之前可能发生了尿素或酸诱导的变性。在酸性pH值下，特别是hFSH的非常快速的解离速率表明离子相互作用对亚基缔合现象有重要贡献。
