• 作者： 張均昌; 陳金珠; 陳行明
• 作者服務機構： 私立高雄醫學院生物化學科
• 中文摘要： 利用CM-Sephadex及DEAE-cellulose管柱層析法，從眼鏡蛇（Taiwan cobra，Naja naja atra）毒中分離出來
  之磷脂?A （Phospholipase A ），以聚丙烯醯胺膠電泳法及免疫電泳法證明它為單一蛋白質。
  磷脂?A 分子中只含有一個組織胺酸（Histidine）殘基，利用p-Bromophenacyl bromide修飾時，酵素的活性完
  全消失，鈣離子對此失活作用具有保護效果。另外，其分子中的三個色胺酸(Tryptophan)殘基被HNB-bromide修飾後
  ，酵素的活性降低至原來之約十分之一，但是這兩種化學修飾衍生物與抗體結合的能力並沒有改變。由這些結果得知，磷
  脂?A 分子中唯一的組織胺酸殘基可能為其酵素活性基，但它與色胺酸殘基一樣，均與抗原決定基無密切關係。
  以磷脂胺A 免疫家兔所得到之抗血清，利用免疫沈澱法及親和層析法精製出來的純化抗體，以木瓜?消化後，再利
  用親和層析法及膠過濾法可分離出Fab單價抗體。這些抗體製品均可以完全抑制磷脂?A 的活性。雖然抑制百分之五十酵
  素活性所需的抗體摩爾數，Fab單價抗體是雙價抗體的兩倍，若以抗體價數作比較，則兩者具有相同之抑制能力。
  測定單價抗體Fab與磷脂?A 反應形成之可溶性複合物的分子量，得知磷脂?A 分子中具有三個抗原決定基。
  i
• 英文摘要： Phospholipase A was isolated from the venom of Taiwan cobra (Naia naia atra) by ion-exchangecnromatograpny on um-Sepltadex and 1)EAEcellulose, followed by gel filtration on Sephadex G -5O. Theenzyme was proved to be electrophoretically and immunochemically homogeneous and was purified 9.5-foldwith an overall yield of 32%. The enzyme activity disappeared completely when single histidine residue in phospholipase A wasmodified with p-bromophenacyl bromide. Calcium ions showed a considerable protection against this inac-tivation. .Approximately 10% residual activity was observed as all three tryptophan residues were alkylatedwith 2-hydroxy-5-nitrobenzyl bromide. These results indicate that histidine residue may be involved in theactive site of phospholipase A . The antigenic activities of both modified derivatives remained intact, in-dicating that histidine as well as tryptophan residues might not be closely related to the antigenicity of theenzyme. The antibody was purified immunospecifically frorp the pooled sera of rabbits which had been hy-perimmunized with phospholipase A . The monovalent antibody, Fab, was isolated from papaindigests ofpurified antibody by affinity chromatography and gel filtration. Each antibody prcparation could inactivatecompletely the phospholipase A activity. The inhibitory activity of divalent antibody seems to be the sameas that of monovalent antibody based on the antibody valence. An estimate of the molecular weight ofsoluble complex formed from Fab with phospholipase A indicates that cobra phospholipase A exhibitsthree antigenic sites per molecule.
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