Okabe N, Fujita E, Tomita K I
Biochim Biophys Acta. 1982 Jan 18;700(2):165-70. doi: 10.1016/0167-4838(82)90093-0.
The denaturation of bovine pancreatic DNAase I (EC 3.1.21.1) by guanidine hydrochloride (GdnHCl) has been investigated with circular dichroism in the presence and absence of 1 mM Ca2+ at the wavelength region of 210-240 nm at 12.25 and 36 degree C. The change of the molar ellipticity at 220 nm by GdnHCl titration showed cooperative transition at each temperature and the midpoints of the titrations occurred near 2 M GdnHCl. At each temperature, the denaturation of DNAase I in the presence of 1 mM Ca2+ occurred a little slowly as compared with that in the absence of Ca2+. This suggests that 1 mM Ca2+ can to some extent stabilize the secondary structure of DNAase I against GdnHCl denaturation. The apparent free energy for the denaturation of DNAase I obtained by GdnHCl titration was calculated as 9.3 +/- 0.3 kcal/mol and 8.9 +/- 0.2 kcal/mol at 25 degree C in the presence and absence of 1 mM Ca2+, respectively. The possible regions for the alpha -helix and beta -structure of DNAase I were predicted from the amino acid sequence by probability calculation of Chou, P.Y. and Fasman, G.D., Adv. Enzymol. 47, 45-148. The characteristic feature is that the NH2-terminal half of DNAase I is rich in beta -structure and the COOH-terminal half contains mainly alpha -helix.
在12.25℃和36℃下，于210 - 240nm波长范围内，利用圆二色性研究了盐酸胍（GdnHCl）对牛胰脱氧核糖核酸酶I（EC 3.1.21.1）的变性作用，实验分别在有和无1 mM Ca2+的条件下进行。GdnHCl滴定引起的220nm处摩尔椭圆率的变化在每个温度下均显示出协同转变，滴定中点出现在接近2 M GdnHCl处。在每个温度下，与无Ca2+时相比，1 mM Ca2+存在时脱氧核糖核酸酶I的变性发生得稍慢一些。这表明1 mM Ca2+在一定程度上可以稳定脱氧核糖核酸酶I的二级结构，使其抵抗GdnHCl变性。通过GdnHCl滴定得到的脱氧核糖核酸酶I变性的表观自由能，在25℃、有1 mM Ca2+存在时计算为9.3±0.3 kcal/mol，无1 mM Ca2+时为8.9±0.2 kcal/mol。根据Chou, P.Y.和Fasman, G.D.在《Adv. Enzymol. 47, 45 - 148》中提出的概率计算方法，从氨基酸序列预测了脱氧核糖核酸酶I的α -螺旋和β -结构的可能区域。其特征在于脱氧核糖核酸酶I的NH2 -末端一半富含β -结构，而COOH -末端一半主要包含α -螺旋。
