使用双检测器池在尺寸排阻高效液相色谱（SEC-HPLC）中同时测量散射光强度和浓度来测定蛋白质的第二维里系数。
Determination of second virial coefficient of proteins using a dual-detector cell for simultaneous measurement of scattered light intensity and concentration in SEC-HPLC.
作者信息
Bajaj Harminder, Sharma Vikas K, Kalonia Devendra S
机构信息
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, USA.
出版信息
Biophys J. 2004 Dec;87(6):4048-55. doi: 10.1529/biophysj.104.048686. Epub 2004 Oct 1.
Abstract
A method is proposed for the measurement of the B22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90 degrees , after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration (ultraviolet) detector is converted to Rayleigh's ratio, Rtheta, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/Rtheta versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature.
摘要
提出了一种在流动模式下测量水溶液中蛋白质B22值的方法，该方法利用一种新制造的双探测器池，在蛋白质从尺寸排阻柱洗脱后，同时测量蛋白质浓度和90度处相应的散射光强度。从光散射探测器和浓度（紫外）探测器获得的色谱图上的每个数据点分别转换为瑞利比Rθ和浓度c。B22值由单次蛋白质进样从这些色谱图获得的一系列浓度生成的德拜图（Kc/Rθ对c）的斜率计算得出。结果表明，该方法能可靠地测定溶菌酶、胰凝乳蛋白酶原和胰凝乳蛋白酶等蛋白质在各种溶液条件下的B22值，与文献报道的值吻合良好。
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