In this study, we purified and characterized flaxseed cysteine protease (FSCP) with strong anticoagulant, antiplatelet, and clot-dissolving properties. The enzyme was purified to homogeneity by a combination of gel permeation and ion-exchange column chromatography techniques. The purity of the enzyme was evaluated by SDS-PAGE, RP-HPLC, and MALDI-TOF. FSCP was observed as a single band of approximately 160 kDa in SDS-PAGE under reducing and non-reducing conditions. The exact molecular mass of FSCP was found to be 168 kDa by MALDI-TOF spectrometry. The CD spectra of FSCP revealed the presence of 25.6% helices, 25.8% turns, and 48% random coils with no beta-sheet structures. FSCP hydrolyzed both casein and gelatin with a specific activity of 3.5 and 4.2 unit/mg min respectively. The proteolytic activity of FSCP was completely abolished by iodoacetic acid (IAA), suggesting FSCP is a cysteine protease. The pH optimum for the proteolytic activity of FSCP was pH 6.0; the temperature optimum was 30°C. FSCP exhibited strong anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) by extending the clotting time from 222 to 1100 s and from 256 to 1210 s, respectively. FSCP degraded human fibrinogen and fibrin clots. The products of fibrinogen degradation by thrombin and FSCP were different. Furthermore, FSCP inhibited aggregation of washed platelets triggered by ADP, epinephrine, thrombin, collagen, arachidonic acid, and platelet activating factor (PAF). FSCP was found to be nontoxic as it did not damage the membrane of red blood cells (RBCs) and did not induce hemorrhage and edema in experimental mice.

中文翻译：

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亚麻籽半胱氨酸蛋白酶具有很强的抗凝、抗血小板和血栓溶解特性

在这项研究中，我们纯化并表征了亚麻籽半胱氨酸蛋白酶 (FSCP)，具有很强的抗凝、抗血小板和溶解凝块的特性。通过凝胶渗透和离子交换柱色谱技术的组合将酶纯化至均质。通过 SDS-PAGE、RP-HPLC 和 MALDI-TOF 评估酶的纯度。在还原和非还原条件下，在 SDS-PAGE 中观察到 FSCP 为大约 160 kDa 的单条带。通过 MALDI-TOF 光谱法发现 FSCP 的确切分子质量为 168 kDa。FSCP 的 CD 光谱显示存在 25.6% 的螺旋、25.8% 的匝数和 48% 的无β-折叠结构的无规卷曲。FSCP 水解酪蛋白和明胶的比活性分别为 3.5 和 4.2 单位/mg min。FSCP 的蛋白水解活性被碘乙酸 (IAA) 完全消除，表明 FSCP 是一种半胱氨酸蛋白酶。FSCP蛋白水解活性的最适pH为6.0；最佳温度为 30°C。通过将凝血时间分别从 222 秒延长至 1100 秒和从 256 秒延长至 1210 秒，FSCP 在富血小板血浆 (PRP) 和贫血小板血浆 (PPP) 中均表现出强大的抗凝作用。FSCP 降解人纤维蛋白原和纤维蛋白凝块。凝血酶和FSCP降解纤维蛋白原的产物不同。此外，FSCP 抑制了由 ADP、肾上腺素、凝血酶、胶原蛋白、花生四烯酸和血小板活化因子 (PAF) 引发的洗涤血小板的聚集。