Kavanagh G M, Clark A H, Ross-Murphy S B
Division of Life Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo, SE1 8WA, London, UK.
Int J Biol Macromol. 2000 Oct 10;28(1):41-50. doi: 10.1016/s0141-8130(00)00144-6.
Heat-set gels and aggregates from beta-lactoglobulin (beta-Lg), one of the major globular proteins from milk, have been studied on a molecular distance scale using negative-staining transmission electron microscopy (TEM), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared spectroscopy (FTIR). The microscopy showed long linear aggregates forming in solutions at pH 2 (and sometimes 2.5) after prolonged heating. While there appeared to be no differences in aggregates formed under these conditions in H(2)O as compared with D(2)O, at all other pH and pD values, and in the presence of added salt, much shorter linear aggregates were formed. These became slightly more extended the further the pH was removed from pI. Wide-angle X-ray diffraction (WAXD) showed a diffuse beta-sheet halo at 2θ=19 degrees in patterns for both dried native and aggregated protein (irrespective of pH) with only a small change (sharpening) of this feature on heat treatment. Solution FTIR spectra, measured at pD=2, 2.5, 3, and 7, during heating, indicated shoulder development at 1612 cm(-1) in the carbonyl-stretching Amide I region diagnostic of a modest increase in intermolecular beta-sheet. In terms of the shoulder size, no distinctions could be made between acid and neutral aggregate structures. At all pHs, beta-lactoglobulin showed only limited secondary and tertiary structural changes in aggregation, in contrast to previous studies of insulin aggregation, where highly ordered crystalline fibrils were indicated. The current work has implications both in structural studies of food biopolymers and in ongoing studies of pathological protein self-assembly in disease states, such as spongiform encephalopathies.
利用负染色透射电子显微镜（TEM）、广角X射线衍射（WAXD）和傅里叶变换红外光谱（FTIR），在分子距离尺度上研究了乳中主要球状蛋白之一β-乳球蛋白（β-Lg）形成的热凝凝胶和聚集体。显微镜观察显示，长时间加热后，在pH 2（有时为2.5）的溶液中形成了长的线性聚集体。虽然在这些条件下，与重水（D₂O）相比，在水（H₂O）中形成的聚集体似乎没有差异，但在所有其他pH和pD值以及添加盐的情况下，形成的线性聚集体要短得多。随着pH值与等电点（pI）的差距增大，这些聚集体会稍微延伸得更长一些。广角X射线衍射（WAXD）显示，干燥的天然蛋白和聚集蛋白的图谱在2θ = 19°处都有一个弥散的β-折叠晕圈（与pH无关），热处理后这一特征只有很小的变化（变锐）。在加热过程中，在pD = 2、2.5、3和7时测量的溶液FTIR光谱表明，在羰基伸缩酰胺I区域1612 cm⁻¹处出现肩峰，这表明分子间β-折叠适度增加。就肩峰大小而言，酸性和中性聚集体结构之间没有区别。与之前对胰岛素聚集的研究不同，在胰岛素聚集中显示出高度有序的结晶纤维，而在所有pH值下，β-乳球蛋白在聚集过程中仅表现出有限的二级和三级结构变化。目前的工作对食品生物聚合物的结构研究以及对疾病状态（如海绵状脑病）中病理性蛋白质自组装的正在进行的研究都有意义。
