Bao Yongbo, Wang Juanjuan, Li Chenghua, Li Peifen, Wang Sufang, Lin Zhihua
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Zhejiang 315100, PR China.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Zhejiang 315100, PR China; School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
Fish Shellfish Immunol. 2016 Apr;51:9-16. doi: 10.1016/j.fsi.2016.02.004. Epub 2016 Feb 12.
The blood clam, Tegillarca granosa, is one of the few bivalve molluscs containing hemoglobin (Hb). In the present study, we purified two types of T. granosa hemoglobin, Tg-HbI and Tg-HbII, using size exclusion chromatography and measured their antibacterial and peroxidase activities. We also tested antibacterial activities of peptides prepared by trypsin digestion of purified Tg-Hb and reversed-phase high-performance liquid chromatography purification. Purified Tg-HbI and Tg-HbII showed antibacterial activity against Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Bacillus firmus, with differences in minimal inhibitory concentrations (MICs), but lacked antibacterial activity against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In contrast, 7 Tg-Hb derived peptides exhibited varying degrees of antibacterial activity against V. alginolyticus (MICs: 12-200 μg/ml), V. parahaemolyticus (11-100 μg/ml) and V. harveyi (1-200 μg/ml). The antibacterial activity of Hb derived peptides was confirmed by fluorescence microscopy. In addition, peroxidase activity was detected in Tg-HbI and Tg-HbII. The results indicated that in addition to functioning as a respiratory protein T. granosa hemoglobins likely play a role in host antibacterial defense probably via a peroxidase activity of native molecules and some internal peptides released from the proteins.
血蚶（Tegillarca granosa）是少数含有血红蛋白（Hb）的双壳贝类软体动物之一。在本研究中，我们使用尺寸排阻色谱法纯化了两种血蚶血红蛋白，即Tg-HbI和Tg-HbII，并测定了它们的抗菌和过氧化物酶活性。我们还测试了通过胰蛋白酶消化纯化的Tg-Hb并经反相高效液相色谱法纯化制备的肽的抗菌活性。纯化的Tg-HbI和Tg-HbII对大肠杆菌、恶臭假单胞菌、枯草芽孢杆菌和坚强芽孢杆菌具有抗菌活性，最小抑菌浓度（MIC）有所不同，但对溶藻弧菌、副溶血性弧菌、哈维氏弧菌和金黄色葡萄球菌没有抗菌活性。相比之下，7种源自Tg-Hb的肽对溶藻弧菌（MIC：12-200μg/ml）、副溶血性弧菌（11-100μg/ml）和哈维氏弧菌（1-200μg/ml）表现出不同程度的抗菌活性。通过荧光显微镜证实了源自Hb的肽的抗菌活性。此外，在Tg-HbI和Tg-HbII中检测到了过氧化物酶活性。结果表明，除了作为呼吸蛋白发挥作用外，血蚶血红蛋白可能通过天然分子和从蛋白质释放的一些内部肽的过氧化物酶活性在宿主抗菌防御中发挥作用。
Zotero 插件