液体核磁共振扩散序谱（DOSY）可以通过测定溶质分子的自扩散系数（Dt）来研究该分子在溶液中的表观分子量（M）.Dt与测试体系和分子本身性质相关，蛋白质体系较为复杂，从而增加了蛋白质自扩散系数（Dt-protein）测定的难度.本文以3-（三甲基硅基）丙磺酸钠（DSS）为内标，以蛋白质分子与DSS自扩散系数的比值（Dr）来表征蛋白质分子在溶液中的表观分子量（Mprotein），该方法降低了缓冲体系对Mprotein的影响，使得Mprotein主要由分子本身的性质决定.在此基础上，测定了不同分子量蛋白质分子相对于DSS的Dr，拟合得到了Dr与Mprotein的相关关系：lgMprotein =-2.6488 lgDr-0.7863，相关系数（R2）为0.997.最后测定了通过大肠杆菌表达纯化得到的SARS冠状病毒主蛋白酶C端结构域（Mpro-C）分子相对于DSS的Dr，并计算出与文献结果一致的Mprotein，进一步验证了拟合公式的准确性和实用性.
李红卫
,
袁志良
,
夏斌
. 扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量[J]. 波谱学杂志, 2018
, 35(3)
: 280
-286
.
DOI: 10.11938/cjmr20182625
Diffusion ordered NMR spectroscopy (DOSY) can be used to determine the apparent molecular weight of proteins (Mprotein) by measuring self-diffusion coefficient (Dt-protein). However, Dt-protein is also affected by the property of buffer solution, making the measurements complicated. In this work, 3-(trimethylsilyl) propane sodium sulfonate (DSS) was used as the internal reference molecule and the diffusion coefficient ratio between the target protein and DSS (Dr) was used to characterize the protein in solution. This method reduced the effects of buffer system on Mprotein determination, yielding more accurate Mprotein measurement. The Dr values of reference proteins with different molecular weight were determined. A correlation curve between Dr and Mprotein was established (lgMprotein=-2.6488 lgDr-0.7863, R2=0.997). Finally, Dr of the C-terminal domain of SARS-CoV main protease (Mpro-C) was determined and to yield its molecular weight. Accuracy and practicability of the fitting curve were demonstrated.
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