全 文 ：菌 物 系 统 21（1）：112~115, 2002
Mycosystema

PURIFICATION AND IDENTIFICATION OF á-AMANITIN AND
PHALLOIDIN FROM AMANITA FULIGINEA*
ZHANG Xiao-Yuan LIANG Song-Ping ZHANG Zhi-Guang
(College of Life Science, Hunan Normal University, Changsha 410081)

ABSTRACT: α-amanitin and phalloidin are cyclic peptide toxins composed of some modified amino
acids distributing mainly in poisonous Amanita mushroom. The application of these toxic peptides in life
science research is extending. In this report, an improved extraction method was employed andα-amanitin
and phalloidin were prepared with reverse-HPLC. The isolated toxins were identified by UV absorbent
spectra and mass spectra. It was showed that the isolated toxins were homogeneous. These procedures can
be used for isolatingα-amanitin and phalloidin from other poisonous mushroom.
KEY WORDS: cyclic peptide toxin, isolation, identification, poisonous mushroom

The great majority of all fatal cases of human mushroom poisoning occur with eating Amanita species,
particularly the green death cap A. phalloides (Faulstich & Thoms, 1994). The mushroom is found frequently
all over Europe and in North America, Africa and Asia (Faulstich & Thomas, 1994; Li et al.,1993). In Hunan
Province, Southern China, however, the toxic mushroom resulting in the fatal casualties is A. fuliginea (Li &
Zhang, 1997), which was first described by Hongo (1953).There are three classes of cyclic peptide toxins in
poisonous Amanita mushrooms including amatoxins (e.g. á-amanitin), phallotoxins(e.g. phalloidin) and
virotoxins, but the facts contributed to fatal casualties are amatoxins (Wieland, 1986). It is of interest to note
that the features of specific inhibition of α-amanitin (á-AMA) on eukaryotic cell RNA polymerase Ⅱ and
specific binding of phalloidin (PHD) on filament actin are useful in molecular biology and cell biology. The
application of amanita toxic peptide in life science research is extending (Zhang et al., 1999). In this paper,
we report the isolation of á-AMA and PHD from A. fuliginea with reversed phase high-performance liquid
chromatography (HPLC) and mass spectrometry.
1 MATERIALS AND METHODS
1.1 Materials
The mushrooms of Amanita fuliginea were collected from Guiyang County, Hunan Province, China in
1997. The fruitbodies were air dried at 40~45℃ and stored at room temperature.

* Supported by National Natural Sciences Foundation of China[国家自然科学基金资助项目 93-(7)-03 and 96C-02-03-07] and
by the Science and Technology Commission of Hunan Province
E-mail: xyzhang@mail.hunnu.edu.cn
Received:2001-05-08, accepted: 2001-06-01
DOI:10.13346/j.mycosystema.2002.01.023
1期 张晓元等：α-鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定 113
1.2 Experimental methods
1.2.1 Extraction for Amanita peptide toxins: The extraction procedure was a modification developed by
Zhang et al.,(1998).The proteins and lipids were removed with chloroform instead of gasline.
1.2.2 Purification for α-AMA and PHD:α-AMA and PHD were isolated by a reversed-phase HPLC. The
HPLC system was Waters 2010 separation module, Waters 486 photodiode array detector. The mobile phases
were employed as Zhang et al. (1998). Separation were performed on a self-packed column with YWG-C18
(300mm×7.5mm I.D.) at 20℃. The flow rate was 2.0 ml.min-1 and the detection wavelength was 295nm.
1.2.3 Identification for α-AMA and PHD: U.V spectrum: The recovery toxins by HPLC were scanned from
200nm to 350nm with Shimadzu UV-2201 spectrometer with distilled water as control. Mass spectrum
analysis: MALDI-TOF MS was carried out with a Bruker Proflex Ⅲ mass spectrometer that was equipped
with a nitrogen laser of 337 nm. The acceleration voltage was set to 20 KV. The spectra data were calibrated
with a Bruker mixed standards (118.09Da, 322.05Da, 622.03Da, 922.01Da and 1521.97Da) externally. The
matrix-α-cyano-4-hydroxycinnamic acid was dissolved in a 0.1% TFA acetonitrile/water(1:2) to a saturated
solution.
2 RESULTS
The reverse-phase HPLC chromatogram for the extraction solution of A. fuliginea fruitbody was given
in fig.1. On the basis of our previous analysis work(Zhang et al., 1998), the retention time ofα-AMA and
Fig.1 The reverse-phase HPLC chromatogram for the extraction solution of
A. fuliginea fruitbody
114 菌 物 系 统 21卷
PHD from A. fuliginea were 17.05 and 30.33 min respectively. á-AMA and PHD have a maximum
characteristic absorbency in 304nm and 290nm respectively, these could be checked in Waters 996
Photodiode Array detector and analyzed by UV spectrum.
á-AMA and PHD are bicyclic peptide composed of some modified amino acids. The systematic name of
á-AMAs Cyclic (L-asparaginyl-trans-4-hydroxy- L-prolyl-(R)-4,5-dihydroxy- L-isoleucyl-6-hydroxy- 2-
mercapto-L-tryptophylglycyl-L-isoleucylglyl-L-cysteinyl)-cyclic-(4→ 8)-sulfide-(R)-S-oxide, and that of
PHD is Cyclic(L-alanyl-D-threonyl-L-cysteinyl-cis-4-hydroxy- L-prolyl-L-akabyl-2-mercapto-L-tryptohyl-
4,5-dihydroxyleucyl) cyclic(3-6) sulfide. It is difficult to identify them by amino acid sequence as usual. Fig.2
shows the result of MALDI-TOF MS analysis ofα-AMA with a mass of 919Da(M + Na+ = 941.4m/z, M +
K+=957.4m/z).So does PHD with a mass of 789Da. These are agreement with Fluka chemical in molecular
masses.
3 DISCUSSIONS
After removing impure proteins and lipids from mushroom extraction solution by chloroform, the
isolation efficiency was improved greatly. As our previous work there were some other toxic peptides such as
â-amanitin(with a RT of 12.2min), phalloin(with a RT of 32.4 min) etc. in poisonous Amanita mushroom,
however there areα-AMA and PHD generally in those Amanita mushroom resulting in the fatal casualties.
It is easy to show that the identification of á-AMA and PHD was conducted by UV spectrum, retention
time in HPLC and mass spectra. á-AMA and PHD, low-molecular weight peptides, have characteristic λmax
because their chromophoric group are 2’-(SOR)6’ -(OH)trp and 2’-(SR)trp respectively. There are nine cyclic
toxic peptides in Amatoxins and seven in Phalltoxins. Owing to their chemical similarity there would be more
than one cyclic peptide in a recovery peak from HPLC elution if the isolation parameters were selected
improperly, thus it can be discriminated by mass spectrum.
[REFEERENCES]
Faulstich H, Thomas R Z, 1994. Amatoxins, In Spoerke DG, Rumack BH(eds.), Handbook of mushroom poisoning:
diagnosis and treatment. U.S.A. CRC Press, Inc., P.233.
Hongo T, 1953. Large fungi of the provinces of Omi and Yamashiro, J Jap Bot, 28: 69~75.
Fig.2 The result of MALDI-TOF MS analysis ofα-AMA with a mass of 919Da
(M + Na+ = 941.4m/z, M + K+=957.4m/z)
1期 张晓元等：α-鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定 115
Li DP, Zhang ZG, 1997. A preliminary assay of several toxic peptides from Amanita fuliginea by HPLC. Life Science
Research, 1: 43~47.
Li JZ, Hu XW, Peng YB, 1993. Large Fungi in Hunan Province. Hunan Normal University Press, 418.
Wieland T, 1986. Peptide of Poisonous Amanita Mushrooms. Springer-Verlag New York Inc, 256.
Zhang XY, Liang SP, Zhang ZG, Chen ZH, 1998. Determination of Toxic Peptides in Amanita virosa and Amanita verna.
Journal of Hygiene Research , 27 :418~420.
Zhang ZG, Zhang XY, Li DP, 1999. The application of amanita toxic peptides in life science research. Journal of Hygiene
Research, 28: 60~63.
á-鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定
张晓元 梁宋平 张志光
（湖南师范大学生命科学院 长沙 410081）

摘 要：á-鹅膏毒（环）肽和二羟鬼笔毒（环）肽是剧毒的鹅膏菌和其它几种致死毒菌中由
一些修饰氨基酸组成的环肽毒素。由于 á-鹅膏毒肽对真核生物的 mRNA合成的专一性抑制和
和二羟鬼笔毒肽对肌动蛋白的专一性束缚，因而它们在分子生物学和细胞学研究中具有重要
应用，对其需求逐步增加。为此，作者使用了一种改良的毒素提取方法，以制备高效液相色
谱从灰花纹鹅膏菌中分离制备 á-鹅膏毒肽和二羟鬼笔毒肽，并通过紫外吸收光谱和质谱进行
鉴定，表明 á-鹅膏毒肽和二羟鬼笔毒肽的分离效果好，纯度高。本方法对其它毒菌中的 á-鹅
膏毒肽和二羟鬼笔毒肽的分离制备具有同样的应用价值。
关键词：环肽毒素， 分离纯化，鉴定，毒菌
中图分类号：Q939.96 文献标识码：A 文章编号：1007-3515（2002）01-0112-0115


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