Backes B J, Rychnovsky S D
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Anal Biochem. 1992 Aug 15;205(1):96-9. doi: 10.1016/0003-2697(92)90584-t.
A quick and simple affinity chromatography method for gauging the interaction of polyene antifungal agents with sterols has been developed. The required affinity columns are prepared from a standard C-18 reverse-phase HPLC column by injecting a measured quantity of sterol under conditions where it is completely retained. After the assay, the sterol is eluted with a less polar solvent and the column reused. By comparing the elution volume of a polyene injected onto the sterol-free column (Ve) with that of the polyene injected onto the sterol-doped column (V), an association constant (Ka) for the polyene-sterol complex was determined. Association constants of different amphotericin B-sterol and pimaricin-sterol complexes were determined and correlated with the polyene's ability to induce membrane permeability and its antifungal properties. This procedure provides a new tool for screening polyene macrolides for antifungal therapy.
已开发出一种快速简便的亲和色谱法，用于测定多烯类抗真菌剂与甾醇的相互作用。所需的亲和柱由标准C-18反相高效液相色谱柱制备，通过在甾醇完全保留的条件下注入一定量的甾醇。测定后，用极性较小的溶剂洗脱甾醇，柱子可重复使用。通过比较注入无甾醇柱（Ve）的多烯的洗脱体积与注入掺杂甾醇柱（V）的多烯的洗脱体积，确定了多烯-甾醇复合物的缔合常数（Ka）。测定了不同两性霉素B-甾醇和匹马霉素-甾醇复合物的缔合常数，并将其与多烯诱导膜通透性的能力及其抗真菌特性相关联。该方法为筛选用于抗真菌治疗的多烯大环内酯类药物提供了一种新工具。
