Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP 202002, India.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP 202002, India.
Int J Biol Macromol. 2016 Oct;91:477-85. doi: 10.1016/j.ijbiomac.2016.05.107. Epub 2016 May 31.
Stem bromelain, a cysteine proteases from Ananas comosus is a widely accepted therapeutic drug with broad medicinal application. It exists as intermediate states at pH 2.0 and 10.0, where it encountered in gastrointestinal tract during adsorption (acidic pH) and in gut epithelium (alkaline pH), respectively. In this study, we monitored the thermal aggregation/amyloid formation of SB at different pH intermediate states. Thermal treatment of stem bromelain at pH 10.0 favors the fibrillation in which the extent of aggregation increases with increase in protein concentration. However, no fibril formation in stem bromelain at pH 2.0 was found at all the concentration used at pH 10.0. The fibril formation was confirmed by various techniques such as turbidity measurements, Rayleigh light scattering, dye binding assays and far UV circular dichroism. The Dynamic light scattering confirmed the formation of aggregates by measuring the hydrodynamic radii pattern. Moreover, microscopic techniques were performed to analyze the morphology of fibrils. The aggregation behavior may be due to variation in number of charged amino acid residues. The less negative charge developed at pH 10.0 may be responsible for aggregation. This work helps to overcome the aggregation related problems of stem bromelain during formulations in pharmaceutical industry.
菠萝蛋白酶是一种来自菠萝的半胱氨酸蛋白酶，是一种被广泛认可的治疗药物，具有广泛的药用应用。它以中间状态存在于 pH 值为 2.0 和 10.0 时，分别在胃肠道吸收时（酸性 pH）和在肠上皮细胞中（碱性 pH）遇到。在这项研究中，我们监测了不同 pH 中间状态下 SB 的热聚集/淀粉样纤维形成。在 pH 10.0 下处理菠萝蛋白酶有利于纤维形成，其中聚集程度随蛋白浓度的增加而增加。然而，在 pH 2.0 下的菠萝蛋白酶根本没有发现纤维形成，即使使用 pH 10.0 下的所有浓度。通过浊度测量、瑞利光散射、染料结合测定和远紫外圆二色性等各种技术证实了纤维形成。动态光散射通过测量水动力半径模式证实了聚集体的形成。此外，还进行了微观技术分析纤维的形态。聚集行为可能是由于带电荷的氨基酸残基数目的变化。在 pH 10.0 时形成的负电荷较少，可能是导致聚集的原因。这项工作有助于克服制药工业中在配方过程中菠萝蛋白酶相关的聚集问题。
