Trichloroacetic acid-induced molten globule state of aminoacylase from pig kidney.
The trichloroacetic acid (TCA)-induced unfolding of aminoacylase was investigated by measurement of aggregation, enzyme activity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate (ANS) binding, circular dichroism, and native polyacrylamide gel electrophoresis. The results showed that TCA caused inactivation and unfolding of aminoacylase. Intrinsic fluorescence results demonstrated that the TCA-induced transition of aminoacylase was characterized by two distinct stages during which the fluorescence emission maxima first redshifted to 338 nm and then blueshifted to 332 nm, close to that of native protein. ANS binding measurements revealed that TCA-denatured aminoacylase had a large hydrophobic area for TCA concentration near 2 mM. Comparison of the relative changes in wavelength shift and in the ANS intensity suggested the formation of a stable molten globule state of aminoacylase with a slightly disrupted tertiary structure and more hydrophobic surface than the native protein. Far-UV circular dichroism results provided further support that TCA induced the formation of two partially folded intermediates each with an enhanced native-like secondary structure. The results collectively suggest that a TCA-induced molten globule state is formed and stabilized during unfolding of aminoacylase and that association of the molten globule state may account for precipitation of the protein when denatured by TCA.
Xu
Zhen
Z
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China.
Xie
Qiang
Q
Zhou
Hai-Meng
HM
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D007668:Kidney / Q000201:enzymology*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013552:Swine; D014238:Trichloroacetic Acid / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D014238:Trichloroacetic Acid; C027132:1-anilino-8-naphthalenesulfonate; D000581:Amidohydrolases; C019672:aminoacylase I