Shamsi Tooba Naz, Parveen Romana, Amir Mohd, Baig Mohd Affan, Qureshi M Irfan, Ali Sher, Fatima Sadaf
Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
PLoS One. 2016 Nov 15;11(11):e0165572. doi: 10.1371/journal.pone.0165572. eCollection 2016.
This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials.
Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy.
ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.
To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.
本研究旨在从具有强大药用特性的植物大蒜中纯化并鉴定蛋白酶抑制剂（PI），并探索其作为植物药的潜力。
采用硫酸铵分级分离和在阴离子交换剂Hi-Trap DEAE柱上进行快速蛋白质液相色谱法纯化大蒜蛋白酶抑制剂（ASPI）。通过SDS-PAGE分析纯化后的蛋白质的纯度和分子量。通过基质辅助激光解吸电离飞行时间串联质谱（MALDI TOF-TOF）确认胰蛋白酶抑制性PI的存在，并通过MASCOT数据库进行分析。进一步研究ASPI在极端pH、温度和化学变性剂条件下的动力学性质和稳定性。通过圆二色（CD）光谱法测定二级结构。
约15 kDa的ASPI抑制胰蛋白酶，在MASCOT数据库中与“截短的库尼茨胰蛋白酶抑制剂（大豆）”匹配。纯化后的ASPI比活性为30376.1371 U/mg，纯度提高了159.92倍，产率约为93%。ASPI在pH 2至12范围内相当稳定，在pH 4至5左右活性下降，表明该蛋白质的pI值在此范围内ASPI会聚集。ASPI在较宽的温度范围（10 - 80°C）内表现出稳定性，但超过80°C后活性下降。此外，洗涤剂、氧化剂和还原剂在不同浓度下会使ASPI活性发生变化。动力学分析显示速度与底物浓度呈S形关系，Vmax为240.8（μM/min），Km值为0.12 μM。ASPI表现出非竞争性抑制，Ki为0.08±0.01 nM。远紫外CD在天然pH下显示2.0%的α-螺旋和51%的β-折叠。
总之，纯化后的约15 kDa ASPI在广泛的pH和温度范围内表现出相当的稳定性。总体而言，纯化倍数增加且产率显著。化学修饰研究表明赖氨酸和色氨酸残基作为活性位点中的主要氨基酸存在。因此，具有胰蛋白酶抑制特性的ASPI有潜力用作非细胞毒性临床药物。
