Wajcman H, Ducrocq R, Riou J, Mathis M, Godart C, Prehu C, Galacteros F
Service de Biochimie, Hôpital Henri Mondor, Créteil, 94010, France.
Anal Biochem. 1996 May 15;237(1):80-7. doi: 10.1006/abio.1996.0204.
Human globin chain analysis provides important information on the genetics and molecular pathophysiology of hemoglobinopathies. We propose using perfusion chromatography on the reversed-phase stationary phase to perform these studies. The technique, herein described, involves a high-velocity flow of the mobile phase through a porous chromatographic stationary phase made of microspheres of poly(styrene-divinylbenzene) having throughpores of 6000-8000 A diameter with short diffusive pores of 500-1000 A diameter connected to them. The composition of fetal hemoglobin (Ggamma:Agamma ratio) is determined, using this method, as an order of magnitude faster than with conventional HPLC. Elution is performed by developing a linear gradient of acetonitrile at a flow rate of 3 ml/min (or more), easily obtained on any HPLC machine. Analyses may be done on samples containing as low as 3.0% Hb F. Results are similar to those obtained with the reference HPLC technique, which uses a C4 column. In addition, reversed-phase perfusion chromatography, using a shallow curvilinear gradient, may help in the characterization of Hb variants. This technique allowed us to discriminate several alpha and beta chain mutants from variants that have closely similar patterns of electrophoretic mobilities.
人类珠蛋白链分析为血红蛋白病的遗传学和分子病理生理学提供了重要信息。我们建议使用反相固定相上的灌注色谱法来进行这些研究。本文所述的技术涉及流动相高速通过由聚（苯乙烯 - 二乙烯基苯）微球制成的多孔色谱固定相，该微球具有直径为6000 - 8000埃的通孔以及与之相连的直径为500 - 1000埃的短扩散孔。使用这种方法测定胎儿血红蛋白的组成（Gγ：Aγ比例），其速度比传统高效液相色谱法快一个数量级。通过以3毫升/分钟（或更高）的流速形成乙腈线性梯度进行洗脱，这在任何高效液相色谱仪上都很容易实现。对于含低至3.0% Hb F的样品即可进行分析。结果与使用C4柱的参考高效液相色谱技术所获得的结果相似。此外，使用浅曲线梯度的反相灌注色谱法可能有助于鉴定血红蛋白变体。该技术使我们能够将几种α和β链突变体与具有非常相似电泳迁移模式的变体区分开来。
