草菇毒蛋白(volvatoxin A)含有兩種成分:Volvatoxin A1及Volvatox-
in A2.兩者分子量各為50 kD 及24 kD.當兩者以1:3 比例混合時具有最大
的溶血活性.Volvatoxin A 含有三種生物活性:第一:具有溶血作用(hemol-
ysis):在濃度2μg/ml 時,將人類 O 型血球完全溶解.第二:對老鼠引發扭
曲反應(writhing reaction).第三:改變實驗動物的心電圖(electrocardi-
ogram,ECG),降低 ST 波及改變 T 波.本實驗就是將 Volvatoxin A2 純化
出來,利用各種不同的水解酵素如:lysyl endopeptidase,S.aureus V8 pr-
otease,chymotrpsin 及化合物溴化氰(cyanogen bromide)進行水解,然後
利用自動胺基酸分析儀(automatic amino acid sequencer)分析.blksq..
blksq.之排列順序,經由蛋白質.blksq..blksq.輿圖(peptide mapping)求
得此 Volvatoxin A2 共199個胺基酸的完全序列.合成可能之Volvatoxin
A2之胺端及.blksq.端胺基酸的 Oligonucleotides 當做 Primers,萃取未
成熟草菇之 cDNA 當做模板,利用鏈聚合.blksq.連鎖反應(polymerase ch-
ain reaction,PCR)的技術,選殖了 Volvatoxin A2 之 cDNA 序列,轉譯成
199個胺基酸再利用電腦分析其二級結構,同時與其它溶血蛋白比較胺基酸
序列以研究其結構與活性的關係.

A hemolysin (Volvatoxin A)was isolated from the edible mushr-
oom,Volvariella volvacea.There are two major components in volv-
atoxin A,volvatoxin A1 (VVA1) and volvatoxin A2 (VVA2).The molec-
ular weight of volvatoxin A1 and volvatoxin A2 were found to be
50 kD and 24 kD,respectively.When the two proteins were mixed
together,the toxicity of volvatoxin A2 was increased and the opti-
mal ratio is 1:3 which is the ratio of the two proteins existing
in volvatoxin A.Volvatoxin A was found to have three major biolo-
gical activities. The first, it was a direct hemolytic action:it
can completely lyse human group O red blood cells at a concentra-
tion of 2μg/ml.Second,it caused a writhing reaction of rats when
administered intraperitoneally.Third,in isolated toad hearts the
toxic protein caused ventricular systolic arrest at a dose of 0.1
mg/ml.In this experiment,VVA2 was digested with the protease such
as lysyl endopeptidase,S.aureus V8 protease and chymotrypsin or
chemically hydrolyzed with cyanogen bromide.The sequence of puri-
fied peptides can be obtained by automatic amino acid sequencer.
The complete amino acid sequence was determined by the peptide
mapping.
Synthetic oligonucleotides corresponding to all possible seq-
uences of N-terminal and C-terminal region of VVA2 were used to
generate VVA2-related sequences using the polymerase chain react-
ion on the cDNAs encoding VVA2.The secondary structure was analy-
zed by the computer.The primary structure of VVA2 was used to
compare with other hemolytic proteins to study it's structure
and function relationships.
A hemolysin (Volvatoxin A)was isolated from the edible mushr-