Dixit Vaishali S, Kumar Ameeta R, Pant Aditi, Khan M Islam
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
Biochem Biophys Res Commun. 2004 Mar 5;315(2):477-84. doi: 10.1016/j.bbrc.2004.01.083.
A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.
通过内在荧光、圆二色性和酶活性测量监测，来自串珠镰刀菌的一种低分子量果胶酸裂解酶在其最适pH 8.5时可被尿素和盐酸胍可逆展开。平衡展开研究得出，以尿素为变性剂时，ΔG(H₂O)为1.741千卡/摩尔，D1/2为2.3M，m值为0.755千卡/(摩尔·M)；以盐酸胍为变性剂时，ΔG(H₂O)为1.927千卡/摩尔，D1/2为1.52M，m值为1.27千卡/(摩尔·M)。果胶酸裂解酶在pH 8.5时的热变性即使在75℃下暴露10分钟后也是可逆的。根据pH值从5.0到8.5的热变性曲线计算出的热力学参数得出ΔCp为0.864千卡/(摩尔·K)。在pH 8.5时，ΔG(25℃)为2.06千卡/摩尔，与从化学变性曲线获得的ΔG(H₂O)值吻合良好。如1-苯胺基萘-8-磺酸（ANS）无法结合果胶酸裂解酶所示，在化学或热变性过程中没有疏水口袋暴露。
