Henrik Wiggers

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays.

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  • Henrik Wiggers
  • J Cheleski, Denmark
  • A Zottis, Denmark
  • G. Olivia, Denmark
  • A. D. Andricopulo, Denmark
  • C. A. Montanari, Denmark
  • The Department of Cardiological Medicine B
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.
Udgivelsesdato: 2007-Nov-1
Original languageEnglish
JournalAnalytical Biochemistry
Volume370
Issue1
Pages (from-to)107-14
Number of pages7
ISSN0003-2697
DOIs
Publication statusPublished - 2007

    Research areas

  • Animals, Calorimetry, Catalysis, Dimethyl Sulfoxide, Enzyme Inhibitors, Glyceraldehyde-3-Phosphate Dehydrogenases, Kinetics, Methanol, Protozoan Proteins, Solvents, Spectrophotometry, Trypanosoma cruzi

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