Xiong W, Chai J, Wu J, Tian M, Lu W, Xu X
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Nov 20;41(11):1657-1663. doi: 10.12122/j.issn.1673-4254.2021.11.09.
To investigate the secondary structure, physicochemical properties and antibacterial activity of Brevinin- 2GHk (BR2GK), an antimicrobial peptide from frog skin, and explore its antibacterial mechanism.
BR2GK was synthesized, purified with high performance liquid chromatography (HPLC) and identified using mass spectrometry. Circular dichroism was used to determine the secondary structure and physicochemical properties of BR2GK. Two-fold dilution method was used to determine the antibacterial activity of BR2GK, and its antibacterial mechanism was explored using laser scanning confocal microscopy (LSCM) and scanning electron microscopy (SEM). The hemolytic activity of BR2GK was analyzed in red blood cells. Isothermal titration calorimetry (ITC) and surface plasmon resonance imaging (SPRi) were employed to detect the binding of BR2GK to lipopolysaccharide (LPS), and the antioxidant activity of BR2GK was evaluated using biochemical kits.
The synthesized BR2GK, with a purity exceeding 95% after purification, had the basic characteristics of cationic antimicrobial peptides. BR2GK consisted mainly of α-helical structure in SDS solution and exhibited a broad-spectrum antibacterial activity. Antibacterial activity assay showed that BR2GK had inhibitory and killing activity against a variety of strains with a minimum inhibitory concentration (MIC) of 2.76 μmol/L against . Observation with LSCM and SEM showed that BR2GK at an active concentration caused bacterial cell membrane damage, cell swelling, contraction, deformation, and massive exudation of intracellular contents without causing hemolysis. ITC showed that the binding affinity K of BR2GK to LPS was 18.2±0.8 μmol/L. The antioxidant test showed that BR2GK was capable of effectively scavenging NO, ABTS and DPPH.
BR2GK, as a broad-spectrum antibacterial peptide with also a strong antioxidant capacity, does not cause hemolysis and is capable of binding to LPS. BR2GK has an important value for future design and synthesis of antimicrobial peptides with stronger antibacterial activity and lower cytotoxicity.
研究蛙皮抗菌肽Brevinin-2GHk（BR2GK）的二级结构、理化性质及抗菌活性，并探讨其抗菌机制。
合成BR2GK，采用高效液相色谱（HPLC）进行纯化，并用质谱进行鉴定。利用圆二色光谱法测定BR2GK的二级结构和理化性质。采用二倍稀释法测定BR2GK的抗菌活性，并用激光扫描共聚焦显微镜（LSCM）和扫描电子显微镜（SEM）探讨其抗菌机制。分析BR2GK在红细胞中的溶血活性。采用等温滴定量热法（ITC）和表面等离子体共振成像（SPRi）检测BR2GK与脂多糖（LPS）的结合，并使用生化试剂盒评估BR2GK的抗氧化活性。
合成的BR2GK经纯化后纯度超过95%，具有阳离子抗菌肽的基本特征。BR2GK在SDS溶液中主要由α-螺旋结构组成，具有广谱抗菌活性。抗菌活性测定表明，BR2GK对多种菌株具有抑制和杀伤活性，对[具体菌株]的最低抑菌浓度（MIC）为2.76 μmol/L。LSCM和SEM观察表明，活性浓度的BR2GK可导致细菌细胞膜损伤、细胞肿胀、收缩、变形以及细胞内内容物大量渗出，且不引起溶血。ITC结果显示，BR2GK与LPS的结合亲和力K为18.2±0.8 μmol/L。抗氧化试验表明，BR2GK能够有效清除NO、ABTS和DPPH。
BR2GK作为一种具有强抗氧化能力的广谱抗菌肽，不引起溶血且能与LPS结合。BR2GK对未来设计和合成具有更强抗菌活性和更低细胞毒性的抗菌肽具有重要价值。
