Role of Zinc (II) in the Refolding of Guanidine Hydrochloride Denatured Bovine Carbonic Anhydrase
Küçük Resim Yok
Tarih
1972
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Yayıncı
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
Circular dichroism, ultraviolet difference spectroscopy, and activity measurements (p-nitrophenyl acetate as substrate) have been employed to study the dénaturation and renaturation of bovine carbonic anhydrase B. This metallo-enzyme is readily denatured by guanidine hydrochloride and refolds spontaneously upon removal of the denaturing condition, recovering essentially full (>95%) enzymatic activity. Denaturation in 3 m guanidine hydrochloride at 25° and pH 6, as judged by circular dichroism and ultraviolet spectroscopy, carries both the native and the apoenzyme from the same initial to the same final conformational state, exposing approximately six buried tryptophan side chains, disrupting interactions of aromatic side chains with dissymmetrical regions, and presumably destroying the specific zinc binding site of the enzyme. The transition between native and denatured conformational states appears thermodynamically reversible with or without Zn(II), although in the absence of the metal it occurs at a lower guanidine hydrochloride concentration (1.5 m vs. 1.0 m midpoint). Renaturation kinetics are complex and imply that intermediate species accumulate during the reaction. Under some conditions (dilution from 4.0 m to 1.0 m guanidine hydrochloride, pH 6, 25°) refolding occurs readily if Zn(II) is present during the initial stages of the reaction, whereas it occurs at an extremely low rate if Zn(II) is added later. This suggests that Zn(II) is bound during the initial steps of folding of the polypeptide chain and thus influences the pathway of the reaction although it does not affect the final conformationál state. © 1972, American Chemical Society. All rights reserved.
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Anahtar Kelimeler
Kaynak
Biochemistry
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Scopus Q Değeri
N/A
Cilt
11
Sayı
7
