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KMID : 0545120190290010114
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Journal of Microbiology and Biotechnology
2019 Volume.29 No. 1 p.114 ~ p.126
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Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril
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Park Se-Ho
Lee Jae-Yeul
Cho Hyun-Nam
Kim Kyoung-Ran
Yang Seun-Ah
Kim Hee-Joon
Jhee Kwang-Hwan
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Abstract
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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.
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KEYWORD
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Homocysteine, cucurbit[7]uril, DTNB, ELISA, human cystathionine γ-lyase
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