Knyazeva Ekaterina L, Grishchenko Valery M, Fadeev Roman S, Akatov Vladimir S, Permyakov Sergei E, Permyakov Eugene A
Institute for Biological Instrumentation of the Russian Academy of Sciences.
Biochemistry. 2008 Dec 9;47(49):13127-37. doi: 10.1021/bi801423s.
A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.
用人乳钙结合蛋白α-乳白蛋白（hLA）与油酸（OA）形成的特定复合物（使用OA预处理的离子交换柱制备，即HAMLET）可在多种肿瘤细胞中触发多种细胞死亡途径。人们已经探索了在溶液条件下制备具有与HAMLET相似的结构和细胞毒性特性的hLA-OA复合物的可能性。该复合物是通过在pH 8.3下用OA滴定hLA直至OA临界胶束浓度而形成的。我们已经表明，复合物的形成强烈依赖于钙、离子强度和温度；确定了最佳条件。通过光谱荧光法估计每个hLA分子不可逆结合的OA分子数量（在将负载OA的制剂对水透析后冻干）取决于温度：17℃时为2.9（天然脱辅基hLA；所得复合物称为LA-OA-17状态），45℃时为9（热变性脱辅基hLA；LA-OA-45）。内在色氨酸荧光测量显示，HAMLET、LA-OA-45和OA饱和蛋白的无钙形式的热稳定性显著降低。不可逆结合的OA不影响蛋白质的钙结合常数。荧光和圆二色性（CD）数据的相图分析表明，OA结合过程涉及几种hLA中间体。估计了无钙hLA的有效假平衡OA缔合常数。无钙hLA的远紫外CD光谱表明，蛋白质的所有OA结合形式的特征是α-螺旋结构含量升高。各种hLA-OA复合物对人表皮样喉癌细胞具有相似的细胞毒性活性。总体而言，LA-OA-45复合物具有与HAMLET非常相似的物理化学、结构和细胞毒性特性。这种类似HAMLET的复合物可以在水溶液中形成这一事实使得其制备过程更加透明和可控，为形成具有特定性质的活性复合物开辟了机会。
