人血红蛋白及其三硝基甲苯加合物的高效液相色谱分离

Separation of Human Hemoglobin and Its Adducts with Trinitrotoluene by High Performance Liquid Chromatography (HPLC)

介绍了采用反相大孔径Ｃ４柱分离血红蛋白（Ｈｂ）的新的高效液相色谱（ＨＰＬＣ）条件，流动相采用乙腈－水－三氟乙酸，在梯度洗脱方式下，对Ｈｂ中的血色素（ｈｅｍｅ）、α－和β－链以及未解离的球蛋白进行了分离，速度快且洗脱完全，并用于三硝基甲苯（ＴＮＴ）－Ｈｂ加合物的分离。结果在体外实验发现了ＴＮＴ－Ｈｂ加合物的形成，其水平为２０４ｐｍｏｌ／ｍｇＨｂ。

The isolation of heme, globin chain (α and β chain) and non-cleavage globin of human hemoglobin (Hb) was achieved by revered phase HPLC on a Vydac large-pore C4 column using a modified gradient between aqueous trifluoroacetic acid (TFA) and trifluoroacetic acid in acetonitrile (A : acetonitrile/water/TFA=20/80/ 0.1, B:acetonitrile/water/TFA=60/40/0.1). The elution started at 35%B, linearly increased to 50%B in 30 min and then increased up to 100%B in 5 min, kept 14min, at flow rate of 1 m L/min. The eluate was monitored by UV detector at 220 nm and collected simultaneously by fraction collector for further determination of carbon-14 label radioactivity. The retention time of heme, α- globin chain, β-globin chain and non-cleavage globin was 14.01, 26. 54, 28. 95 and 36. 34 min, respectively. This method was applied to study [14C] trinitrotoluene (TNT)-Hb adducts. The column recovery of [14C] TNT-Hb was about 100%. The results showed that the formation of TNT adducts with Hb was found in vitro, which had a level of 204 p mol/mg Hb.