CRIBI Biotechnology Centre, University of Padua,Viale G. Colombo 3, 35121 Padua, Italy.
Biochemistry. 2010 Oct 5;49(39):8658-67. doi: 10.1021/bi1012832. Epub 2010 Sep 9.
α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4-5 protein molecules that bind 68-85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu.
α-乳白蛋白（LA）与油酸（OA）形成复合物，据报道该复合物能诱导肿瘤细胞的选择性死亡。然而，这种复合物能广泛杀死多种肿瘤细胞系的机制在很大程度上仍然未知。难以合理化 LA/OA 复合物的细胞毒性作用可能是由于蛋白质与脂肪酸之间相互作用的分子方面仍了解甚少，特别是关于蛋白质的寡聚状态和复合物中 OA 相对于蛋白质的实际摩尔比。在这里，通过动态光散射测量和透射电子显微镜检查了 LA 对 OA 水溶液的添加的影响。在蛋白质添加后，相当不溶性的 OA 的聚集状态发生了显著变化，形成了更多水溶性和更小的脂肪酸聚集体。LA 和过量 OA 的混合物形成了高分子量复合物，可以通过尺寸排阻色谱法分离，并且对 Jurkat 细胞显示出细胞毒性。根据凝胶过滤数据、戊二醛交联实验和 OA 滴定，我们评估了分离的 LA/OA 复合物由 4-5 个蛋白质分子组成，这些蛋白质分子结合 68-85 个 OA 分子。复合物中的蛋白质采用类球蛋白样构象，并且通过圆二色性测量和有限的蛋白水解实验表明，它主要通过其α-螺旋结构域与脂肪酸相互作用。总的来说，我们将我们和以前的数据解释为表明 LA/OA 复合物的细胞毒性是由于蛋白质部分显著提高了细胞毒性 OA 的水溶性的影响所致，因此，蛋白质/ OA 复合物主要可以作为在生理环境中携带毒性脂肪酸的载体。
