Mozersky S M, Caldwell K D, Jones S B, Maleeff B E, Barford R A
Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, Pennsylvania 19118.
Anal Biochem. 1988 Jul;172(1):113-23. doi: 10.1016/0003-2697(88)90419-8.
Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.
沉降场流分级法（sed-FFF）被证明是分析多种亚细胞颗粒制剂的一种有价值的方法。本文描述了这种相对较新的分离方法的基本原理。分离基于颗粒在质量和/或密度上的差异。与色谱法一样，该方法涉及将在分离室流出物上进行的在线或离线测量与洗脱（保留）时间相关联。在这项工作中，监测流出物在254、280和/或320nm处的吸光度；对收集的级分进行蛋白质含量、总ATP酶活性和/或标记酶活性测定，并在适当情况下通过电子显微镜检查。发现254和320nm处吸光度的比值可提供亚细胞颗粒部分分辨率的灵敏测量方法。通过sed-FFF检查了含有玉米根所有亚细胞颗粒（不包括细胞核、细胞壁和核糖体）的制剂及其富含线粒体、微粒体、高尔基体膜或质膜的级分。亚细胞颗粒似乎在很大程度上保持完整。观察到的所有颗粒质量均小于2×10¹¹g/mol。所有制剂在有效质量分布方面都非常不均匀。这部分归因于起源细胞器的异质性。在微粒体制剂中，未保留峰中存在低密度、中密度和高密度成分；保留区域中高密度颗粒相对较多。质膜制剂的有效颗粒质量分布非常宽。这些观察结果表明，除了其分析用途外，sed-FFF可能被证明对某些亚细胞颗粒制剂的微量制备分级有用。sed-FFF和密度梯度离心可作为互补方法使用。
