尖吻蝮蛇毒抗血小板聚集活性蛋白的纯化和性质研究

目的 分离纯化尖吻蝮蛇毒中抗血小板聚集活性蛋白agacutegrin,并研究其生化性质.方法依次用Uno Q、UnoS离子交换色谱和反相高效液相色谱分离得到单一组分agacutegrin,用比浊法和MTT法检测其体外抗血小板聚集和抗肿瘤细胞生长活性.用电泳和Edman降解法测定了agacutegrin相对分子质量和N端氨基酸序列.结果和结论Agacutegrin为碱性蛋白,pI为8.04,SDS-PAGE方法测得其相对分子质量为26.59 kDa.Agacutegrin体外具有抗ADP和胶原诱导的血小板聚集活性,IC50分别为153.1 nmol·L-1和214.4 nmol·L-1.此外,agacutegrin还具有较广泛的体外抑制肿瘤细胞生长活性,ID50为18～35 μg·mL-1.Agacutegrin与蛇毒中的C-型凝集素类似蛋白家族成员N端序列高度同源,表明它可能为1个新的C-型凝集素类似蛋白.

Novel Anti-platelet Aggregation Protein from Agkistrodon acutus: Isolation and Characterization

AIM: To investigate the characteristics of a novel anti-platelet protein, agacutegrin, which was purified from Agkistrodon acutus. METHOD: Sequenced Uno Q, Uno S and RP-HPLC were employed in the purification of agacutegrin. Anti-platelet aggregation and anti-cancer activities in vitro were tested by turbidimetric and MTT methods. Molecular mass and sequence character were determined by SDS-PAGE and Edman degradation, respectively. RESULTS AND CONCLUSION: Agacutegrin was a basic protein with pI 8.04. Its molecular weight was estimated as 26.59 kDa by SDS-PAGE. Agacutegrin inhibited the aggregation of human platelet induced by ADP and collagen with IC50 at 153.1 nmol·L-1 and 214.4 nmol·L-1, respectively. In addition, agacutegrin can effectively inhibit various tumor cells proliferation in vitro with ID50 among 18～35 μg·mL-1. Agacutegrin shared high identity with proteins from C-type lectin family of snake venom, which indicated that agacutegrin was a new member of the C-type lectin family.