Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c: stabilization of a native-like folded intermediate

Ahmad, Atta; Madhusudanan, K. P.; Bhakuni, Vinod

doi:10.1016/s0167-4838(00)00069-8

Cited by 29 publications

(28 citation statements)

References 33 publications

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“…It is also known that TFA has denaturing properties [19]. For example, it was demonstrated that TFA (concentrations around 0.1%) induces extensive unfolding of cytochrome c characterised by a significant breakdown of the secondary and tertiary structure of the protein [20]. Such an effect was not observed in the present study for IFN-␣2b in presence of ACN and TFA.…”

Section: Discussioncontrasting

confidence: 65%

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

Luykx

Goerdayal

Dingemanse

et al. 2005

Journal of Chromatography B

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Section: Discussioncontrasting

confidence: 65%

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

Luykx

Goerdayal

Dingemanse

et al. 2005

Journal of Chromatography B

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“…In the 1960s, Charles Tanford18 proposed that TCA forces protein to precipitate by sequestering the protein‐bound water. Subsequently, several other studies suggested that the acidic nature of TCA is important for the conformational changes that trigger protein precipitation 19–21. However, the mechanism by which TCA precipitates proteins is not clearly understood.…”

Section: Introductionmentioning

confidence: 99%

Trichloroacetic acid‐induced protein precipitation involves the reversible association of a stable partially structured intermediate

et al. 2009

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Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2-trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA-induced protein precipitation reaction. TCA-induced protein precipitation curves are U-shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1-anilino-8-napthalene sulfonate (ANS) and size-exclusion chromatography, obtained using acidic fibroblast growth factor (aFGF), show that a stable ''molten globule-like'' partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea-induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. H- 15N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the b-strands at the N-and C-terminal ends (of aFGF) are disrupted in the trichloroacetate-induced ''MG-like'' state. The results of the study clearly demonstrate that TCA-induced protein precipitation occurs due to the reversible association of the ''MG-like'' partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome.

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“…For example, proteins that rely on buried salt bridges are typically among those that aggregate and precipitate upon transition to the A state (57) . The aggregation and precipitation of the A state can also depend on the anions in solution (1,65,66,73) . Interestingly, because of the infl uence of anions on A state stability, not all acids are capable of quantitatively precipitating such proteins (58) .…”

Section: Basic Principles Of the Approachmentioning

confidence: 99%

Large‐Scale Identification of Intrinsically Disordered Proteins

Uversky

Cortese

Tompa

et al. 2010

Instrumental Analysis of Intrinsically Disordered Proteins

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23A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment were also shown experimentally to be totally disordered. A near 100,000 -fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble Escherichia coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver -stained two -dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) gels loaded with 10 μ g of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than threefold higher yield. A substantial yield of unprecipitated protein was ABSTRACT

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Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c: stabilization of a native-like folded intermediate

Cited by 29 publications

References 33 publications

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

Trichloroacetic acid‐induced protein precipitation involves the reversible association of a stable partially structured intermediate

Large‐Scale Identification of Intrinsically Disordered Proteins

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