School of Chemistry, University of Hyderabad, Hyderabad, 500046, India.
Biochemistry (Mosc). 2015 Mar;80(3):332-42. doi: 10.1134/S0006297915030086.
A novel defensin-like antifungal peptide (Tf-AFP) with molecular mass of 10.3 kDa was isolated from seeds of Trigonella foenum-graecum (fenugreek) by ammonium sulfate precipitation, cation-exchange, gel-filtration, hydrophobic chromatography, and RP-HPLC. Mass spectroscopic analysis revealed the intact mass of the purified antifungal peptide as 10321.5 Da and high similarity to plant defensins and other antifungal proteins in database search. 2D-PAGE showed pI value to be 8.8 and absence of isoforms. Isolated Tf-AFP inhibited growth of fungal species such as Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. The antifungal activity was inhibited in the presence of 50 mM NaCl. Circular dichroism analysis demonstrated that the protein is rich in β-sheet structure and highly stable over a wide range of temperatures. Surprisingly, reduction of disulfide bridges and chemical denaturation did not produce large changes in secondary structure as judged by circular dichroism as well as by fluorescence spectroscopy.
一种新型防御素样抗真菌肽（Tf-AFP），分子量为 10.3 kDa，从葫芦巴（胡芦巴）种子中通过硫酸铵沉淀、阳离子交换、凝胶过滤、疏水色谱和反相高效液相色谱法分离得到。质谱分析显示，纯化的抗真菌肽的完整质量为 10321.5 Da，与数据库搜索中的植物防御素和其他抗真菌蛋白高度相似。2D-PAGE 显示等电点为 8.8，不存在同工型。分离的 Tf-AFP 抑制了尖孢镰刀菌、茄病镰刀菌和立枯丝核菌等真菌的生长。在存在 50 mM NaCl 的情况下，抗真菌活性受到抑制。圆二色性分析表明，该蛋白富含β-折叠结构，在很宽的温度范围内高度稳定。令人惊讶的是，还原二硫键和化学变性并没有像圆二色性和荧光光谱分析所判断的那样，使二级结构发生大的变化。
