扩散序谱(DOSY)实验测定缓冲体系中蛋白质表观分子量  

Determination of Apparent Protein Molecular Weight in Solution by Diffusion Ordered NMR Spectroscopy

作　　者：李红卫 袁志良 夏斌[1,2,3] LI Hong-wei;YUAN Zhi-liang;XIA Bin(Beijing Nuclear Magnetic Resonance Center,Peking University,Beijing 100871,China;College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China;College of Life Sciences,Peking University,Beijing 100871,China)

机构地区：[1]北京大学北京核磁共振中心,北京100871 [2]北京大学化学与分子工程学院,北京100871 [3]北京大学生命科学学院,北京100871

出　　处：《波谱学杂志》2018年第3期280-286,407,408,409,410,共11页Chinese Journal of Magnetic Resonance

基　　金：国家重点研发计划资助项目(2016YFA0501203);国家自然科学基金青年科学基金资助项目(21705005)

摘　　要：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(D_t)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(D_(t-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(D_r)来表征蛋白质分子在溶液中的表观分子量(M_(protein)),该方法降低了缓冲体系对M_(protein)的影响,使得M_(protein)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D_r,拟合得到了D_r与M_(protein)的相关关系:lgM_(protein)=-2.6488 lgD_r-0.7863,相关系数(R^2)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(M^(pro)-C)分子相对于DSS的D_r,并计算出与文献结果一致的M_(protein),进一步验证了拟合公式的准确性和实用性.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,R^2=0.997).Finally,Dr of the C-terminal domain of SARS-CoV main protease(M^pro-C)was determined and to yield its molecular weight.Accuracy and practicability of the fitting curve were demonstrated.

关 键 词：液体核磁共振(liquid-state NMR) 表观分子量 扩散序谱(DOSY) 缓冲体系 脉冲梯度场 

分 类 号：O482.53[理学—固体物理]