Mass spectrometric identification of pyroglutamic acid in peptides following selective hydrolysis

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• 作者：Amit Kumar M ; al ; Padmanabhan Balaram
• 关键词：Chagas disease ; Isothermal titration calorimetry ; Enzyme kinetics ; Organic solvents
• 刊名：Analytical Biochemistry
• 出版年：2007
• 出版时间：1 November 2007
• 年：2007
• 卷：370
• 期：1
• 页码：118-120
• 全文大小：175 K

文摘

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.