Basilico N, Pagani E, Monti D, Olliaro P, Taramelli D
Istituto di Microbiologia Medica, Università di Milano.
J Antimicrob Chemother. 1998 Jul;42(1):55-60. doi: 10.1093/jac/42.1.55.
The malaria parasite metabolizes haemoglobin and detoxifies the resulting haem by polymerizing it to form haemozoin (malaria pigment). A polymer identical to haemozoin, beta-haematin, can be obtained in vitro from haematin at acidic pH. Quinoline-containing anti-malarials (e.g. chloroquine) inhibit the formation of either polymer. Haem polymerization is an essential and unique pharmacological target. To identify molecules with haem polymerization inhibitory activity (HPIA) and quantify their potency, we developed a simple, inexpensive, quantitative in-vitro spectrophotometric microassay of haem polymerization. The assay uses 96-well U-bottomed polystyrene microplates and requires 24 h and a microplate reader. The relative amounts of polymerized and unpolymerized haematin are determined, based on solubility in DMSO, by measuring absorbance at 405 nm in the presence of test compounds as compared with untreated controls. The final product (a solid precipitate of polymerized haematin) was validated using infrared spectroscopy and the assay proved reproducible; in this assay, activity could be partly predicted based on the compound's chemical structure. Both water-soluble and water-insoluble compounds can be quantified by this method. Although the throughput of this assay is lower than that of radiometric methods, the assay is easier to set up and cheaper, and avoids the problems related to radioactive waste disposal.
疟原虫代谢血红蛋白，并通过将其聚合形成疟色素（疟原虫色素）来解毒所产生的血红素。一种与疟色素相同的聚合物β-血红素可以在体外从酸性pH值的血红素中获得。含喹啉的抗疟药（如氯喹）会抑制这两种聚合物的形成。血红素聚合是一个重要且独特的药理学靶点。为了鉴定具有血红素聚合抑制活性（HPIA）的分子并量化其效力，我们开发了一种简单、廉价的体外分光光度法定量微量测定血红素聚合的方法。该测定使用96孔U型底聚苯乙烯微量板，需要24小时和一台酶标仪。通过在存在测试化合物的情况下与未处理的对照相比，在405nm处测量吸光度，基于在二甲基亚砜中的溶解度来确定聚合和未聚合血红素的相对量。使用红外光谱对最终产物（聚合血红素的固体沉淀物）进行了验证，并且该测定被证明具有可重复性；在该测定中，活性可以部分地基于化合物的化学结构来预测。水溶性和水不溶性化合物都可以通过这种方法进行量化。尽管该测定法的通量低于放射性方法，但该测定法更易于设置且成本更低，并且避免了与放射性废物处理相关的问题。
