Tobler S A, Sherman N E, Fernandez E J
Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, P.O. Box 400741, Charlottesville, VA 22904-4741.
Biotechnol Bioeng. 2000;71(3):194-207. doi: 10.1002/1097-0290(2000)71:3<194::aid-bit1009>3.0.co;2-q.
We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the identification of the conformers present.
我们利用电喷雾电离质谱（ESI-MS）和氢-氘交换（HX）来检测盐诱导沉淀过程中蛋清溶菌酶的去折叠情况。将氘代溶菌酶溶解在pH 2.16的质子化缓冲液中，并用硫酸铵、氯化钠和硫氰酸钾进行沉淀。ESI-MS用于检测由于溶剂质子取代氘核导致的溶菌酶质量差异，从而深入了解标记实验过程中蛋白质的构象变化历程。硫酸铵和氯化钠沉淀不会使溶菌酶去折叠，这与促溶剂盐已知的稳定作用一致。硫氰酸钾是一种强变性剂，在0.2 M时是一种有效的沉淀剂，但也会破坏溶菌酶结构，并导致形成沉淀组分，这些沉淀组分在不使用化学变性剂的情况下不易重新溶解于水溶液中。用1.0 M硫氰酸盐沉淀导致更快的去折叠速率和更多的不溶性沉淀。去折叠动力学是双相的，在数小时后呈现出一个缓慢阶段，这可能反映了溶菌酶在沉淀状态下去折叠的倾向较小。硫氰酸盐沉淀的ESI-MS谱中的双峰质量分布表明该系统中溶菌酶存在两种状态，一种是天然状态，另一种是类似熔球的部分去折叠状态。对不溶性沉淀的ESI-MS分析表明，它们主要由已去折叠的蛋白质分子组成。对低蛋白浓度的KSCN溶液中溶菌酶的HX行为研究证实了盐对蛋白质结构的去稳定作用，即使几乎没有固相存在时也是如此。HX/ESI-MS结果为这样一个系统中沉淀和变性相结合的机制提供了深入了解，包括获得定量的动力学和稳定性信息以及鉴定存在的构象体。
