Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570 006, India.
Thromb Res. 2010 Mar;125(3):e100-5. doi: 10.1016/j.thromres.2009.10.002. Epub 2009 Oct 24.
Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.
Pergularain e I 是一种半胱氨酸蛋白酶，具有凝血酶样活性，从 Pergularia extensa 的乳胶中通过离子交换层析进行纯化。其均一性通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、天然聚丙烯酰胺凝胶电泳和反相高效液相色谱（RP-HPLC）进行表征。基质辅助激光解吸电离飞行时间（MALDI-TOF）测定的 pergularain e I 的分子量为 23.356 kDa，N 端序列为 L-P-H-D-V-E。Pergularain e I 是一种糖蛋白，含有约 20%的碳水化合物。Pergularain e I 占总蛋白的 6.7%，比活为 9.5 单位/毫克/分钟，纯度提高了 2.11 倍。碘乙酸（IAA）完全抑制 pergularain e I 的蛋白水解活性。Pergularain e I 对柠檬酸血浆和纤维蛋白原具有类似于凝血酶的促凝活性。Pergularain e I 以浓度和时间依赖的方式增加纤维蛋白原溶液的吸光度。在 10μg 浓度下，孵育 10 分钟内吸光度达到 0.48。当使用 0.2 NIH 单位的凝血酶时，观察到类似的吸光度。Pergularain e I 的凝血酶样活性是由于纤维蛋白原 Aα和 Bβ链的选择性水解，而γ链被观察到不易水解。在 5 分钟孵育时间内，由于 pergularain e I 的水解，从纤维蛋白原释放的两个肽片段的分子量分别为 1537.21 和 1553.29，与纤维蛋白肽 A 的 16 个氨基酸序列和纤维蛋白肽 B 的 14 个氨基酸序列的分子量非常吻合。延长纤维蛋白原与 pergularain e I 的孵育时间会释放出额外的肽，并且释放肽的分子量的序列比较表明 pergularain e I 特异性地在精氨酸残基后水解。
