Park Se-Ho, Lee Jae-Yeul, Cho Hyun-Nam, Kim Kyoung-Ran, Yang Seun-Ah, Kim Hee-Joon, Jhee Kwang-Hwan
Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea.
Institute of Natural Science, Keimyung University, Daegu 42601, Republic of Korea.
J Microbiol Biotechnol. 2019 Jan 28;29(1):114-126. doi: 10.4014/jmb.1811.11029.
This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH₂CH₂SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.
本文介绍了三种确定葫芦[7]脲（CB[7]）与同型半胱氨酸（Hcy）主客体络合作用的方法。将Hcy和半胱氨酸（Cys）与CB[7]预孵育后，使用埃尔曼试剂（DTNB）检测Hcy和Cys。只有Cys与DTNB反应，而Hcy的颜色变化延迟。这表明Hcy的-SH基团被埋在CB[7]内部。人胱硫醚γ-裂合酶（hCGL）在将Hcy和CB[7]预孵育后降低了Hcy的降解水平。这些结果表明，Hcy-CB[7]络合物的形成减少了游离Hcy的量。将CB[7]与Hcy预孵育后，抗Hcy单克隆抗体的免疫信号显著降低。酶联免疫吸附测定（ELISA）结果还表明，Hcy的乙硫醇基团（-CH₂CH₂SH）作为抗Hcy单克隆抗体的一个表位，被CB[7]的空腔所阻断。总体而言，CB[7]可通过与Hcy选择性结合而充当主体，但不与Cys结合。使用埃尔曼方法计算出CB[7]的半络合形成浓度为58.2 nmol，使用hCGL测定法为97.9 nmol，使用单克隆抗体为87.7 nmol。Hcy和Cys对CB[7]主体的不同结合能力可能为测定血浆或血清中Hcy浓度提供一种简单而有用的方法。
