Wiggers H J, Cheleski J, Zottis A, Oliva G, Andricopulo A D, Montanari C A
Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil.
Anal Biochem. 2007 Nov 1;370(1):107-14. doi: 10.1016/j.ab.2007.06.042. Epub 2007 Jul 6.
In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.
在药物研发项目中，二甲基亚砜（DMSO）是一种广泛用于生化分析的标准溶剂。尽管在有机溶剂存在的情况下对酶进行了广泛的使用和研究，但对于某些酶来说，有机溶剂的影响尚不清楚。在进行生化筛选时，大分子靶点可能会受到不同溶剂的影响，以至于构象变化会扰乱其活性位点结构，同时伴随着活性的显著变化。为了解决这个问题，在这项工作中，我们研究了两种有机溶剂二甲基亚砜（DMSO）和甲醇（MeOH）在等温滴定量热法（ITC）动力学分析中对克氏锥虫甘油醛-3-磷酸脱氢酶（GAPDH）催化反应的影响。此前尚未研究过溶剂对克氏锥虫GAPDH的影响。结果表明，该酶在此受到有机溶剂含量的影响，甲醇含量高达5.0%，二甲基亚砜含量高达7.5%。结果表明，当使用ITC实验在DMSO（5%，v/v）存在的情况下测定GAPDH时，该酶的活性比未添加共溶剂的GAPDH高出约两倍。当甲醇（5%，v/v）作为共溶剂时，GAPDH活性高出六倍。有机溶剂对米氏酶-底物复合物形成的有利影响确保了生物学分析的一致性、蛋白质的结构完整性以及测量时间内的可重复性。还通过标准分光光度法对反应进行了动力学监测，以确定克氏锥虫GAPDH在用于筛选潜在抑制剂时的行为表现。
