Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c: stabilization of a native-like folded intermediate(1).
A systematic investigation of trichloroacetic acid (TCA) and trifluoroacetic acid (TFA)-induced equilibrium unfolding of native horse cytochrome c has been carried out using a combination of optical spectroscopy and electrospray ionization mass spectroscopy (ESI MS). In the presence of an increasing concentration of TCA the native cytochrome c does not undergo significant unfolding but stabilization of a partially folded intermediate is observed. This TCA-induced partially folding intermediate of cytochrome c had an enhanced secondary structure and slightly disrupted tertiary structure compared to native protein and undergoes extensive unfolding in the presence of TFA. However, in the presence of an increasing concentration of TFA, cytochrome c was found to undergo extensive unfolding characterized by a significant breakdown of the secondary and tertiary structure of protein. The TFA-unfolded cytochrome c was found to undergo folding in the presence of TCA and low guanidine hydrochloride (GdmCl) resulting in the stabilization of the partially folded intermediate. The effectiveness of TCA as compared to TFA in the stabilization of intermediates was further supported by the observation that low concentrations of TCA were found to induce refolding of HCl-denatured cytochrome c whereas, under similar concentrations of acid, no significant effect on the unfolded structure of protein was observed in the presence of TFA. ESI MS studies indicated that the trichloroacetate anion has a greater affinity for cytochrome c compared to trifluoroacetate anion, which might be the reason for the stabilization of the native-like folded intermediate during TCA-induced denaturation of cytochrome c as compared to extensive unfolding observed in the presence of TFA.
Ahmad
A
A
Division of Membrane Biology, Central Drug Research Institute, Lucknow, India.
Madhusudanan
K P
KP
Bhakuni
V
V
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D013058:Mass Spectrometry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D014238:Trichloroacetic Acid / Q000737:chemistry* / Q000378:metabolism
D003574:Cytochrome c Group; D014238:Trichloroacetic Acid