Callahan J W, Shankaran P, Khalil M, Gerrie J
Can J Biochem. 1978 Sep;56(9):885-91. doi: 10.1139/o78-137.
Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A-Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 degrees C. The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.
鞘磷脂酶从人胎盘中纯化出来，纯化倍数约为1700倍。该过程的主要步骤包括在伴刀豆球蛋白A - 琼脂糖、琼脂糖6B和羧甲基 - 琼脂糖（CM - 琼脂糖）上进行色谱分离。最终制剂在4℃储存时至少可稳定3个月。发现该酶在CM - 琼脂糖和等电聚焦上具有异质性。纯化过程中使用的大多数缓冲液中存在的 Triton X - 100似乎是导致异质性的部分原因。当用Bio Beads处理去除 Triton X - 100时，异质性降低。然而，去除去污剂也会导致酶活性丧失，且再添加 Triton X - 100无法恢复这种活性丧失。数据表明鞘磷脂酶具有高度疏水性，其稳定性和电聚焦行为均受与非离子去污剂相互作用的影响。
