Zhang H J, Sheng X R, Pan X M, Zhou J M
Institute of Biophysics, Academia Sinica, Beijing, 100101, China.
Biochem Biophys Res Commun. 1997 Sep 18;238(2):382-6. doi: 10.1006/bbrc.1997.7301.
The unfolding of adenylate kinase in urea or guanidine hydrochloride solutions was measured by UV absorbance at 287 nm, circular dichroism at 222 nm and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence. At concentrations less than 1.8 M of urea, the secondary and tertiary structures of AK were not noticeably perturbed. In contrast, the activity of the enzyme underwent significant changes, increasing about 1.6-fold when the urea concentration was increased to 1 M. The enzyme activity then decreased with further increases of the urea concentration. We also observed that the kinetics of ANS binding to AK by fluorescence was biphasic. The fast phase completed within the dead-time of the stopped-flow apparatus used, while the slow phase ended in about 10 minutes. The slow phase fluorescence rate constants increased from 0.0073 s-1 in the absence of denaturants to 0.0100 s-1 (about 1.4-fold) at 1 M urea and then decreased at higher urea concentrations. Similar results were obtained when guanidine hydrochloride was used as a denaturant. The change of the enzyme activity coincided with that of the rate of ANS binding during denaturation by low concentration of denaturants, suggesting that the activation of AK by denaturants may be due to the increasing conformational flexibility at its active site.
通过测量287nm处的紫外吸光度、222nm处的圆二色性以及8-苯胺基-1-萘磺酸（ANS）荧光，来测定腺苷酸激酶在尿素或盐酸胍溶液中的去折叠情况。在尿素浓度低于1.8M时，AK的二级和三级结构未受到明显扰动。相比之下，酶的活性发生了显著变化，当尿素浓度增加到1M时，活性增加约1.6倍。随后，随着尿素浓度的进一步增加，酶活性下降。我们还观察到，通过荧光测定的ANS与AK结合的动力学是双相的。快速相在所用停流装置的死时间内完成，而慢速相在大约10分钟内结束。慢速相荧光速率常数从无变性剂时的0.0073 s-1增加到1M尿素时的0.0100 s-1（约1.4倍），然后在更高的尿素浓度下下降。当使用盐酸胍作为变性剂时，也得到了类似的结果。在低浓度变性剂变性过程中，酶活性的变化与ANS结合速率的变化一致，这表明变性剂对AK的激活可能是由于其活性位点构象灵活性的增加。
