Abstract：
The structural stability and therapeutic activity of Stem Bromelain (BM) have been explored by unravelling the interaction of stem BM in presence of two different types of anionic surfactants namely, bile salts, NaC and NaDC and the conventional anionic surfactants, SDDS and SDBS, below, at and above the critical micelle concentration (cmc) in aqueous phosphate buffer of pH 7. Different physicochemical parameters like, surface excess (Γcmc), minimum area of surfactants at air water interface (Amin) etc. are calculated from tensiometry both in absence and presence of BM. Several inflection points (C1, C2 and C3) have been found in tensiometry profile of surfactants in presence of BM due to the conformational change of BM assisted by surfactants. Similar observation also found in isothermal titration calorimetry (ITC) profiles where the enthalpy of micellization (ΔH0obs) of surfactants in absence and presence of BM have calculated. Further, steady state absorption and fluorescence spectra monitoring the tryptophan (Trp) emission of free BM and in presence of all the surfactants at three different temperatures (288.15 K, 298.15 K, and 308.15 K) reveal the nature of fluorescence quenching of BM in presence of bile salts/surfactants. Time resolved fluorescence studies at room temperature also support to determine the several quenching parameters. The binding constant (Kb) of BM with all the surfactants and free energy of binding (∆G0 of bile salts/surfactants with BM at different temperatures have been calculated exploiting steady state fluorescence technique. It is observed that, the binding of NaC with BM is greater as compared to other surfactants while Stern-Volmer quenching constant (KSV) is found greater in presence of SDBS as compared with others which supports the surface tension and ITC data with the fact that surface activity of surfactant(s) is decreasing with the binding of the surfactants at the core or binding pocket of BM. Circular Dichroism (CD) study shows the stability of secondary structure of BM in presence of NaC and NaDC below C3, while BM lost its structural stability even at very low surfactant concentration of SDDS and SDBS which also supports the more involvement of bile salts in binding rather than surfactants. The molecular docking studies have also been substantiated for better understanding the several experimental investigations interaction of BM with the bile salts/surfactants.
摘要：
茎菠萝蛋白酶（BM）的结构稳定性和治疗活性已经通过解开茎BM在两种不同类型的阴离子表面活性剂的存在下的相互作用进行了探索，即，胆汁盐，NaC和NaDC与常规阴离子表面活性剂,SDDS和SDBS,下面,在pH为7的磷酸盐水溶液中的临界胶束浓度（cmc）以上。不同的物理化学参数，表面过量(Γcmc)，空气-水界面表面活性剂的最小面积(Amin)等。从在不存在和存在BM的情况下的张力法计算。由于表面活性剂辅助的BM构象变化，在BM存在下，表面活性剂的张力计分布中发现了几个拐点（C1，C2和C3）。在等温滴定量热法（ITC）曲线中也发现了类似的观察结果，其中计算了在不存在和存在BM的情况下表面活性剂的胶束化焓（ΔH0obs）。Further,稳态吸收和荧光光谱监测游离BM的色氨酸（Trp）发射，并且在三种不同温度下存在所有表面活性剂（288.15K，298.15K,和308.15K）揭示了在胆汁盐/表面活性剂存在下BM荧光猝灭的性质。室温下的时间分辨荧光研究也支持确定几个猝灭参数。利用稳态荧光技术计算了BM与所有表面活性剂的结合常数（Kb）和结合自由能（胆盐/表面活性剂在不同温度下与BM的G0。据观察,与其他表面活性剂相比,NaC与BM的结合更大,而与支持表面张力和ITC数据的其他表面活性剂相比,在SDBS存在下发现Stern-Volmer猝灭常数(KSV)更大,事实是表面活性剂的表面活性随着表面活性剂在BM的核心或结合袋处的结合而降低。圆二色性（CD）研究表明，在低于C3的NaC和NaDC存在下，BM的二级结构的稳定性，而BM即使在非常低的SDDS和SDBS表面活性剂浓度下也失去了其结构稳定性，这也支持胆汁盐更多地参与结合而不是表面活性剂。分子对接研究也已得到证实，可以更好地理解BM与胆汁盐/表面活性剂的相互作用。
