Majumdar Sourav, Sarmah Biplob, Gogoi Debananda, Banerjee Subhamoy, Ghosh Siddhartha S, Banerjee Subham, Chattopadhyay Pronobesh, Mukherjee Ashis K
Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Science, Tezpur University, Tezpur 784028, Assam, India.
Department Biotechnology, Indian Institute of Technology, Guwahati 781039, Assam, India.
Biochimie. 2014 Aug;103:50-60. doi: 10.1016/j.biochi.2014.04.002. Epub 2014 Apr 13.
In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 μmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.
在本研究中，已报道了从短短芽孢杆菌FF02B菌株中纯化得到的纤维蛋白溶解丝氨酸蛋白酶——短链溶栓酶的生化和药理学特性。还对其溶栓效力进行了评估。通过SDS-PAGE和MALDI-TOF-MS测定，这种单体蛋白酶的分子量分别为55 kDa和56043 Da。在分析研究中，发现短链溶栓酶的N端序列被封闭；然而，肽质量指纹图谱和氨基酸组成分析表明，短链溶栓酶与内肽酶在催化三联体中含有丝氨酸方面具有相似性。丝氨酸蛋白酶抑制剂可降低短链溶栓酶的催化（纤维蛋白溶解）活性，这一观察结果证实了这一发现。短链溶栓酶的二级结构由30.6%的α螺旋和69.4%的无规卷曲组成。短链溶栓酶对纤溶酶的显色底物的Km和Vmax值分别为0.39 mM和14.3 μmol/min。短链溶栓酶在pH 7.4和37℃时表现出最佳的纤维蛋白溶解活性，对球蛋白、酪蛋白和纤维蛋白原表现出微弱的水解活性。短链溶栓酶的抗凝效力与低分子量肝素/抗凝血酶III和华法林相当。在短链溶栓酶、纤溶酶和链激酶这三种酶中，短链溶栓酶的纤维蛋白溶解活性和体外溶栓效力被发现是 superior。RP-HPLC和SDS-PAGE分析表明，短链溶栓酶和纤溶酶对纤维蛋白的降解模式相似，表明前者是一种类似纤溶酶的纤维蛋白溶解丝氨酸蛋白酶。短链溶栓酶在HT29和HeLa细胞上未表现出体外细胞毒性或溶血活性。在剂量为10 mg/kg时，短链溶栓酶对Wistar品系白化大鼠未表现出致死性或毒性。短链溶栓酶不抑制因子Xa，其抗凝作用机制与凝血酶的酶促裂解有关。短链溶栓酶的综合特性表明其在基于肽的心血管药物开发中的治疗潜力。
