Stachurska Karolina, Marcisz Urszula, Długosz Maciej, Antosiewicz Jan M
Biophysics Division, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5 Strasse, 02-093 Warsaw, Poland.
ACS Omega. 2022 Jun 28;7(27):23782-23789. doi: 10.1021/acsomega.2c02438. eCollection 2022 Jul 12.
We recorded the far- and near-UV circular dichroism (CD) spectra of solutions of α-chymotrypsin and sodium dodecyl sulfate (SDS) with the final surfactant concentration significantly above the critical micellization concentration. Solutions were prepared using three different procedures. The reference procedure was to mix the chymotrypsin solution with the SDS solution once, immediately achieving the final SDS concentration. In alternative procedures, the protein solutions initially contained some SDS and were mixed with pure SDS solutions at a concentration to provide the same final surfactant as the reference mixing. We demonstrate that the supplementation to the selected final concentration of SDS of the pure chymotrypsin solution leads to different CD spectra than the supplementation to this final concentration of SDS in the chymotrypsin solution containing a small concentration of a few millimolar SDS. These differences disappear when the initial concentration of SDS in the protein solution, which we then supplement to the indicated final concentration, is higher. This suggests the irreversibility of the processes caused by the addition of SDS to chymotrypsin and the influence of the initial amount of this surfactant on the processes occurring with its further addition to the solution. For quantitative analysis of far-UV CD spectra in terms of populations of protein secondary structure elements, we used four well-established software packages. All programs consistently indicate that the observed differences in the far-UV CD spectra can be explained by the differences in the increase in the population of helical forms in chymotrypsin under the influence of SDS.
我们记录了α-胰凝乳蛋白酶和十二烷基硫酸钠（SDS）溶液在表面活性剂最终浓度显著高于临界胶束浓度时的远紫外和近紫外圆二色性（CD）光谱。使用三种不同的程序制备溶液。参考程序是将胰凝乳蛋白酶溶液与SDS溶液混合一次，立即达到最终SDS浓度。在替代程序中，蛋白质溶液最初含有一些SDS，并与纯SDS溶液混合，其浓度能提供与参考混合相同的最终表面活性剂。我们证明，向选定的最终浓度的SDS中添加纯胰凝乳蛋白酶溶液会导致与向含有几毫摩尔低浓度SDS的胰凝乳蛋白酶溶液中添加该最终浓度的SDS不同的CD光谱。当我们随后将蛋白质溶液中SDS的初始浓度补充到指定的最终浓度时，如果该初始浓度较高，这些差异就会消失。这表明向胰凝乳蛋白酶中添加SDS所引起的过程是不可逆的，并且这种表面活性剂的初始量会影响其进一步添加到溶液中时所发生的过程。为了根据蛋白质二级结构元件的数量对远紫外CD光谱进行定量分析，我们使用了四个成熟的软件包。所有程序一致表明，在SDS的影响下，胰凝乳蛋白酶中螺旋形式数量增加的差异可以解释远紫外CD光谱中观察到的差异。
