Srinivasulu S, Rao A G
Department of Protein Technology, Central Food Technological Research Institute, Mysore, India.
Biochim Biophys Acta. 1996 May 23;1294(2):115-20. doi: 10.1016/0167-4838(95)00263-4.
The unfolding of lipoxygenase-1 by urea and guanidine hydrochloride has been followed at the optimum pH of enzyme activity. The unfolding of lipoxygenase-1 by urea or guanidine hydrochloride was characterized by equilibrium transition curves for different parameters like (i) enzyme activity, (ii) change in ellipticity values at 222 nm, and (iii) relative fluorescence intensity at 332 nm could not be superimposed. The transition curves displayed more than one plateau region suggesting the presence of stable intermediates during unfolding. At urea concentrations less than 1 M there was no significant loss in activity although loss in secondary structure was approximately 20%. At 4.0 M urea concentration there was complete loss of activity with a midpoint concentration of 2.5 M urea. The loss in secondary structure was biphasic. The first transition had a midpoint concentration of 1.2 M, while the second transition which was complete at 8.0 M urea had a midpoint concentration of 3.5 M urea. The changes in relative fluorescence intensity and shift in emission maximum were complete at 8.0 M urea. The Stern-Volmer constant for acrylamide and potassium iodide did not change at urea concentrations less than 4 M and then at higher concentrations increased. The reactivity of sulfhydryl groups to Ellman's reagent increased during the course of unfolding. The kinetics of unfolding supported the presence of stable intermediates during unfolding. The unfolding was irreversible and complex because of the multidomain nature. The apparent irreversibility could be related to aggregation during unfolding which precluded the determination of thermodynamic parameters.
在脂氧合酶-1的酶活性最佳pH值条件下，研究了尿素和盐酸胍对其的去折叠过程。尿素或盐酸胍诱导脂氧合酶-1去折叠的过程通过不同参数的平衡转变曲线来表征，如（i）酶活性、（ii）222nm处椭圆率值的变化以及（iii）332nm处的相对荧光强度，这些曲线无法重叠。转变曲线显示出不止一个平台区域，表明去折叠过程中存在稳定的中间体。在尿素浓度低于1M时，尽管二级结构损失约20%，但活性没有显著损失。在尿素浓度为4.0M时，活性完全丧失，中点浓度为2.5M尿素。二级结构的损失是双相的。第一个转变的中点浓度为1.2M，而在8.0M尿素时完成的第二个转变的中点浓度为3.5M尿素。相对荧光强度的变化和发射最大值的位移在8.0M尿素时完成。在尿素浓度低于4M时，丙烯酰胺和碘化钾的斯特恩-沃尔默常数不变，然后在较高浓度时增加。在去折叠过程中，巯基对埃尔曼试剂的反应性增加。去折叠动力学支持去折叠过程中存在稳定中间体。由于其多结构域性质，去折叠是不可逆且复杂的。明显的不可逆性可能与去折叠过程中的聚集有关，这妨碍了热力学参数的测定。
