Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.
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. (1)H-(15)N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the beta-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.
Rajalingam
Dakshinamurthy
D
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Loftis
Charles
C
Xu
Jiashou J
JJ
Kumar
Thallapuranam Krishnaswamy S
TK
D055598:Chemical Phenomena; D011232:Chemical Precipitation; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D014238:Trichloroacetic Acid / Q000737:chemistry*
D011506:Proteins; D016220:Fibroblast Growth Factor 1; D014238:Trichloroacetic Acid