Ellman's-reagent-mediated regeneration of trypanothione in situ:: substrate economical microplate and time-dependent inhibition assays for trypanothione reductase
Ellman's-reagent-mediated regeneration of trypanothione in situ:: substrate economical microplate and time-dependent inhibition assays for trypanothione reductase
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DOI：
10.1042/bj20021298
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发表时间：
2003-02-01
影响因子：
4.1
通讯作者：
Fairlamb, AH
中科院分区：
文献类型：
--
作者：
Hamilton, CJ;Saravanamuthu, A;Fairlamb, AH
锥硫酮还原酶（TryR）是参与锥虫属和利什曼原虫属寄生虫氧化应激管理的关键酶，通过将小分子量二硫化物锥硫酮（T[S]（2））还原为其二巯基衍生物二氢锥硫酮（T[SH]（2）），有助于维持细胞内还原环境。TryR抑制研究目前受到天然酶底物T[S]高昂成本的影响（2）。这种成本在时间依赖性和高通量抑制测定中特别显著。在本研究中，我们报告了一个协议，大大降低了这种测定所需的基板数量。这是通过将测定与化学氧化剂5，5 '-二硫代双-（2-硝基苯甲酸）（DTNB）偶联来实现的，DTNB可以快速将T[SH]产物再氧化回二硫化物底物T[S]（2），从而保持恒定的底物浓度并避免由于底物耗尽而偏离速率线性。这使得能够开发用于经典和时间依赖性TryR抑制的连续微孔板测定法，其中使用通常在数秒内完全消耗的最小底物浓度（< 1 μ M，与底物K-m值30 μ M相比），线性反应速率可以维持60分钟或更长时间。以这种方式，基板要求降低了几个数量级。利用这些程序还描述了一种新的时间依赖性抑制剂cis-3-oxo-8，9 b-bis-（N-1-acrylamidospermidyl）- 1，2，3，4，4a，9 b-hexahydrobenzofuran（PK 43）的表征。
Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S](2)) to its di-thiol derivative dihydrotrypanothione (T[SH](2)). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S](2). Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH], product back into the disulphide substrate T[S](2), thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60 min or more using minimal substrate concentrations (< 1 muM, compared with a substrate K-m value of 30 muM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis-3-oxo-8,9b-bis-(N-1-acrylamidospermidyl)- 1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.