A Novel Myotoxin from the Venom of Trimeresurus mucrosquamatus

Abstract

A novel myotoxin, designated TMPB, was purified from the venom of Trimeresurus mucrosquamatus by Sephadex G-100 superfine gel chromatography and fast protein liquid chromatography (FPLC). The N-terminal sequence of 24 amino acid residues was determined by protein sequencer. The sequence similarities between TMPB and other two phospholipase A(2) (PLA2s) previously purified from the same venom were 41.7% and 54.2%, respectively, but TMPB showed no detectable PLA(2) hydrolytic activity. Its molecular weight was estimated to be 16 000 by reducing SDS-PAGE and isoelectric point was determined to be 9.2 by isoelectric focusing electrophoresis. TMPB exhibited strong myotoxicity and platelet aggregation inhibiting activity, and the two activities could all be inhibited by heparin.

Full text

ISSN 05829879
生物化学与生物物理学报
A CTA BIOC HIM I CA et BIOP HYS ICA S IN ICA 1999 315483488 CN 311300Q
ReceivedF ebruary 11 1999 A cceptedAp ril 141999
Corresponding aut hor Tel868715194279 Fax 86871
5191823 emailx utr 163 net
A Novel Myotoxin from the Venom of Trimeresurus mucrosquamatus
XU T ianRui WANG WanYu M ENG Q ingXiong HUANG YuHui 
LU Q iuM ing an d X IONG YuLiang 
Kun mi ng Inst it ute of Zoology t he Chinese Academy of S ciences K unming 650223 Chi na 
Abstract  A novel myotoxin design ated T MPB w as purified f rom the v enom of Tri meresurus
mu crosquamatus by Sephadex G100 superfine gel chromatography and f ast protein liquid
chromatography FPLCT he Nterminal sequence of 24 amino acid residues was determined by protein
sequencer The sequence similarities bet ween T M P B and other t wo phospholipase A2PLA2spreviously
purified from the same venom w ere 41 7and 54 2 respectively but T MPB showed no detectable
PLA2hy drolyt ic a ctivit y Its molecular weight w as estimated to be 16 000 by reducing SDSPAGE and
isoelectric point w as deter mined to be 9 2 by isoelectric focusin g electrophoresis T MPB ex hibite d st ron g
my otoxi cit y and platelet agg reg ation in hibitin g activ ity and the two activities could all be inhibited by
heparin 
Key Words  M yotoxin phospholipase A2platelet Tr imeresurus mu crosquamatus venom
M yot oxins proteins that induce skeletal muscle
necrosis are divided into two types1Type I
includes sm all basic polypeptides such as peptide c
2
and my otox in 
3that were isolated from the venom
of ratt lesnakes Ty pe II includes larger proteins that
have t he primary structures of ph ospholipase A 2T he
PLA2myotoxins are the main compo nent of
myotoxins identified in snake venom f rom virtually
every family and genus examined4T he PLA 2

myotoxins are divided into three groups1Group I
includes the presynaptic neurotoxin s w ith PLA2
hydrolytic activity such as c roto x in
5group II
includes the nonneurotoxic P LA2s wi th P LA2
activity such as myo toxin I6an d a myotoxin from
Vipera russel li venom7g roup I I I includes
myotoxin PLA 2s w hich exhibit very low or no
detectable P LA2h yd roly tic a ct iv ity such as a
myotoxin f rom B oth rops n um m ifer venom8and
myotoxin II from Bothrops moojeni venom9
M yonecrosis is a common symptom of victim bit ten
by snakes of Trimeresurus genus t herefor e the
investigation of the myotoxins in Tr imeresu rus
m ucrosqua matus is helpful fo r the treatment of sn ake
bite 
He parin is an impo rt ant r egu lato ry f actor t o
PLA2w hich bin ds to PLA 2noncovalently and can
affe ct t he hy droly tic activities10pharmacological
activities11of PLA 2Thus the ef fects of heparin on
a certain P LA2myotoxin should be examined 
In snake venom t here are numerous proteins
w ith P LA2activity some of t hem affect platelet
agg regatio n
1214So i t is in teresting to ex amine
w hether and how a cert ain PLA2affects platelet
agg regatio nUp to date there is no r epo rt a bout
how heparin affects the platelet aggregation function
of P LA2In t his study we purified and partially
sequenced a basic my oto xin TM PBf rom the venom
of TMucrosquamatus and investigated its platelet
agg regatio n in hibitin g activi ty We also studied the
pha rma co lo gical effe ct s of heparin o n TM P B 
1Materials and Methods
11Materials
The crude ly ophilized venom of T
m ucrosqu am at us w as obtained f rom Ku nming
In stitu te of Zoology th e Chinese Academy of
S cien ce K unming Ch ina The mice used to
investigate myo toxicity were Kunming species t he
platelets of J apanese big ear rabbits were used to
deter mine platelet agg reg ation T he Sephadex G100
superfine gel Resource S cation exchange column and
f ast p ro tein liquid c hroma togr aphy F PL C sy stem
were purchased from Pharm acia Pisc at aw ay N J 
USATh e 476A protein sequencer came from
Ap plied B iosyste m In c Foster Ci ty CA US
AHeparin 10 20 kDand ADP were obtained
from Sigma Chemical Co S t Louis MO US

AAll o t her reagen ts used we re of a naly t ical
g rade 
12Isolation
The Tmucrosquamatus venom 05 g w as
dissolved in 0 05 molL sodiu m phosphate buff er plus
01 molL sodium chlo ride pH 6 2and applied to
Sephadex G100 superfine column 3 cm 100 cm
equilibrated wit h the same buff er Elution w as
perfo rmed using equ ilibrating buffer at a f low rate of
12 5 ml per h our 
The fractions containing myotoxicity f rom
Sephadex G10 0 colum n w ere pooled lyophilized and
dialyzed ag ainst 0 03 molL sodium phosphate buffer
pH 7 5 then applied to Resource S colum n 18
cm 20 cmin the FPLC syst em Elution w as done
w it h a linear gradien t of sodium chloride 01 mol
Lin th e same buffer at a flow rate of 8 ml per
minute 
13Electrophoresis
Reducing sodium dodecyl sulfatepoly acrylam ide
gel electrophoresis SDSPAGEw as c ar ried out on
12 5gels according to the method of Laemmli15
Using t he Pharmacia PhastSystem isoelectric
focusing w as performed w ith a pH gradient from 3 to
10 according to the methods recommended by
PhastGe l IE F M edia 
14Nterminal sequencing
U sin g Edm an degr ad at io n me thod T he N
termin us was sequenced by a 476A pro tein sequencer
from A pplied B iosystem Inc 
15PLA2activity
The PLA2activi ty w as me asu red using sy nthetic
dipalmitoyl Lphosphatidylcholine as subst rate pH
75 at 37  as described by Liu et al14
Enzymatically released f atty acid w as tit rated w ith 4
mmolL NaOH The unit of enzyme activity w as
exp ressed as mol of f atty acid released min per mg
protein 
16Myotoxicity
Twelve male mice 20 25 gw er e divided in
tw o gr ou ps In group A each mo use w as injected
w it h 40 g TMPB in 100 l saline into the left
femo ral muscles In group B control each mouse
w as in jected wit h 100 l saline Af ter 9 h
postinjection half of the mice were sacrificed and af ter
20 h the rest w ere all sacrificed T he femo ral muscles
w ere subsequently removed and fixed in Bouin s
solut ion dehydrated t hrough a series of et hanol of
different concentrations and finally embedded in w ax 
Sections of t he muscles were stained wit h
hem atoxyline and eosin H&E
To investigate the in hibiting effects of heparin 
six mice w ere each injected w ith 40 g TM PB 40
g heparin incubated in 100 l saline solutio n f o r 12
min at 3 7 T hen these mice w ere treated as
described above 
17Platelet aggregation inhibiting activity
Platelet aggregation was measured by t he
turbidimetric meth od using w ashed platelets T he
machine used was a SH93 Intelligent Blood
Agg rega tion and C oagu la tion Te ster Shanghai
Biochemical Instrument F actory Shanghai China
In order to avoid the effects of thrombin in plasma
we p repared w ashed platelets by t he f ollowing
metho d Platelet rich plasma PRPw as obt ained by
centrif uging citrated rabbit blood at 200 gfo r 5 m in
and then P RP w as centrifuged at 900 gfor 10 min 
The supernatant w as discarded and the platelets w ere
w ashed twice and resuspended in Ty rode solution 
Platelet count w as adjusted to 300 000 l
The washed platelets 200 lwere incubated
w ith 1 0 l sample at 37 for 1 min ADP w ith final
concentration of 6 mmolL w as added to initiate
platelet agg regation and the aggregation w as
recorded fo r 5 mi n Sa mple 1 h eparin 04g
sam ple 2 saline solution sample 3 T MPB 04g
heparin 04gincubated in 10 l saline for 12
minsample 4 TM PB 04g
2Results
21Isolation and Electrophores is
As show n in Fig 1A Tmucrosquamat us
venom applied to a Sephadex G10 0 superfine colum n
revealed 7 peaks T he peak 6 possessing the most
poten t myotoxicity w as then applied to a Resource S
column in F PLC sy stem Fig 1B T he b ig g est
peak ex hibitin g my otoxi ci ty w as homo geneous on
bot h reducing SDSPAGE and isoelectric focusing
electrophoresis Th e molecu lar weigh t w as estim ated
to be 16 000 and the isoelect ric point w as estimated
to be 9 2see Fig 2
22The Nterminal sequence
The homogenous my otoxin w as applied to 476A
protein sequencer and t he Nterminal sequence of 24
amino acids was determined Comparing w ith t he
protein se quences in data base of Sw issProt and
NCBI w e fou nd th at th e Ntermin al sequen ce w as a
typical P LA2sequence and named the myotoxin
T M PB 
The Nterminal sequence similarities between
T M PB and ot her tw o P LA2s previously purified from
the same venom were 41 7and 54 2
respectively14 16T he sequence similarities between
TM PB and ot her tw o my otoxins from the venom of
Trimeresurus genus were 45 8and 41 7
respectively17 18The sequen cing result and t he
comparison w ith o ther snake PLA2s from
Trimeresurus genus w ere show n in Table 1 
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AC T A B IOCH IM ICA et BIO P HYSI CA S IN IC A Vol 31No 5

T able 1 Nt erminal amin o acid sequences o f T M PB and severa l P LA 2s from the venom of Tr imeresurus genus
PLA2source Nterm inal sequence Similarity 
Tm ucrosq uamat us AN L L Q F R KM I K K MT G K E P I L S YA T Y 100
Tmucrosquamat us BS L I E L G KM I F Q E T G KN P VK N Y G L Y 41 7
Tm ucrosquama tus CN LWQ F E NM I M K V AK K S G I L S Y S A Y 542
Tflavoviri dis S L V Q LWK M I F Q E T G K EA AK N Y G L Y 45 8
Tgramineus S V I E L G KM I F Q E T G KN P A T S YG L Y 41 7
Th e iden tical amino acids we re show n in bold le tte rs a nd t he nonidentical amino acids w er e showe d in nor mal lett er sTm u crosquam atus A
TM P BTmucrosquamat us B
14Tm ucrosquam a tus C
16Tf lavovi rid is17Tg ram ineus18
Fig 1C hr omato graph y pa ttern s in purify ing T M PB
from the venom of Tri m eresuru s mucrosquamat us
AC hroma tog raphy of c rude venom o n a Sephadex G100
superfine gel column BChro mat ography of myotoxicity peak
f rom S ephadex G100 superfine gel column on a Resource S
cation exchang e colum n in F PLC system 
23PLA2activity
TMPB showed no detectable PLA 2hy dro ly tic
activity 
24Myotoxicity
M acroscopic observation show ed th at th e limbs
injected wi th T M PB were noticeably swollen and
turgid Hemorrhage and edema were very obvious
surrounding the site of injection at 9t h h our A t 20th
hour the affected tissues ap peared very dark and
hard and the hemorrhage w as evident u pon
dissection 
Ligh t microscopy at 9th hour after T M PB
injection paraffin sections revealed an obvious local
intercellular edema most myofibrils sh ow ed a m ore
hyaline and homogenous pattern Fig 3B
comp ar ed w ith the con trol Fig 3A  most of
myofibrils appeared to be in t he init ial stages of
degenera tio n A sign of inflamm ation w as seen as
neut rop hils arou nd h yaline my ofibr ils Fig 3B 
At 20th hour af ter T M PB injection th e affected
myofibrils appeared an amorphous mass of
disorganized and disrupted myofibrils contained in
basal lami na and t here w as e x ten sive hemo rrh ag e in
fo rm o f ex tr avasated e ry th rocytes w ithin t he
connective t issue N eut rophil infilt ration was very
obvious Fig 3C Microscopic examination of t he
tissues injected w ith heparin treated TM PB for 20
hou r show ed essentia lly no rmal m yofi brils although a
few n ecrotic myofib rils were surrounded by
neutrophils and hemorrhage could be seen in the
connective tissue Fig 3D T his suggested
heparin could inhibit the myo toxicity of T M PB 
Fig 2Reducing SDSPAG E and isoelectric f ocusing electro
phoresi s of T M P B
ASDSPAG E 1m arker 2TM P B sampleT he molecular w eight
w as estima ted to be 16 000 BIsoelectric fo cusing elect ropho resis1
ma rker 2T M P B sample The iso electric po int w as estimated to be 92
25Platelet aggregation inhibiting activity
When the samples were mixed wi th w ashed
platelets TM PB could reduce th e rate and ex tent of
platelet aggregation induced by ADP When treated
w ith heparin the in hibiting activity of TM PB was
markedly reduced To exclude the effects of heparin
on platelet agg rega tio n heparin was also used as a
485Sep  19 99 XU T ianRui et a l A N ovel M yot ox in f rom t he Ven om of Tr im eresu rus mucrosquamatus

Fig 3Light micrographs of mouse skeletal muscles
AS keletal muscles afte r injection of 100 l physiological solution fo r 20 h no rmal controlThe myo fib rils ap pe ared
norma l BS keletal mu scles a fter injection o f T MP B for 9 h T he myofibrils appeared a hyaline and homogeneous pat tern 
lots of m yof ibrils w ere sur rounded by ne utro phils arrow heads a f ew myof ibrils w e re necrotic NIntercellular edema
wa s ve ry obv ious CSke letal muscles after i nje ction of T M P B f or 20 h Ne crotic my ofibr ilsNrevealed an am orphous
mass of disorg anized and disrupted my ofibrils contai ned in basal lam inaex ten sive hem orr hage Hand neut ro phils
arrowheadsalso sho wn in t his section DSkeletal muscles after injection of TM PB treated by he pari n f or 20 h 
M yof ibrils a ppeared essent ially no rm altho ugh f ew necrotic myof ibrils w ere surr ounded by neut rophils headsand
h em orr hage Hcould be seen in t he tissues
Fig 4Effe cts of T M PB on ADPindu ced p latele t
ag gregation in rabbit w ashed platelets
1heparin con trol2saline contr ol3T M PB t reated by
heparin 4T M PB 
control We found that heparin did no t effect platelet
agg reg at io n a t th is concent ratio n Fig 4
3Discussion
Chen et al19isolat ed a neuroto x ic PL A2
m ole cula r w eigh t 1 5 000f rom TMucrosqua
mat us venom It exhibited PLA2hydrolytic activity
and showed some myotoxicity TM P B molecular
weigh t 16 000 pI 9 2 however sh ow s no
detectable P LA2hyd rolytic activity and exhibited
strong myot oxicity We searched Medline  S wiss
modeline  Protein Data Bankand G ene Bankin
internet but w e f ound no protein sequence identical
t o T M PB So T M P B i s a novel my oto xin i sola ted
from TM ucrosquama tu s venom T he Nterminal
sequence similarities betw een TM PB and other four
PLA2s from the venom of Tr imeresu r us genu s are
41 7 54 2 45 8and 41 7
respectively14 161 8There are 5 identical amino acid
residues among TM PB and oth er four P LA2s f rom t he
venom of Trimeresuru s genus see Table 1T M PB
exhibit s no detectable P LA2hy droly tic a ct ivity so it
needs fur ther data to ascertain T MPB s PLA 2
486 
AC T A B IOCH IM ICA et BIO P HYSI CA S IN IC A Vol 31No 5

identity 
From Fig 3B it is clear that most myofibrils
revealed a hyaline and homogeneous state and we
think t hat these myofibrils are in the stage betw een
hypercontracting stage and initial stage of disruption 
The neutrophils around the degenerated myofibrils
suggest that neutrophils may play an important role in
myonecrosis 
TMPB exhibits no detectable PLA2activity but
its myot oxicity and platelet agg regation inhibiting
activity are ob vious these suggest i ts myotoxicity and
platelet ag g reg at ion inhibiti ng activi ty are
independent of P LA2hy drolyt ic act iv it y 
F urt hermo re the myotoxicity and platelet
aggreg ation inhibiting activity can all be inhibited by
heparin so we infer th at the myo toxicity site and
platelet aggregation inhibiting site of T M P B may be
the same or overlapping w ith each o ther 
He parin is an impo rt ant r egu lato ry f actor t o
PLA2it binds to PLA2no nco valen tly and af fects the
hydrolytic activities and pharmacological activities of
PLA210 11There were tw o distinct opinions 
howeverabout how heparin binds to P LA2Some
scientists proved t hat heparin interacted w ith t he N
termin us of P LA2the in terfacial r ecog nitio n siteand
prevented PLA2from binding t o substrates10w hile
o thers f ound that heparin bound to PLA2at
continuous st retches of basic amino acids near the C
termin us and formed an inactive acidbase
complex11Because heparin is sulfated
polysaccharides composed of repeating disaccharide
units its longi tudinal axis is long enough to cover the
Nterminus and Cterminus of PLA 2w e th ink t hat
heparin may bind PLA2both on Nterminus and C
termin usBo th Nterminus and Ctermin us are all
critical to pharmacological activities of P LA2411
therefore w e th ink t his may be a reason w hy heparin
is a powerful and multivalent inhibitor of TM PB 
Acknowledgements  W e g rate fully acknow ledge
Dr Zh ang Y  M r Jin Y  Wen P J Z hu S W
and Li D Sin our laboratory for t heir valuable
suggests and technique support s 
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487Sep  19 99 XU T ianRui et a l A N ovel M yot ox in f rom t he Ven om of Tr im eresu rus mucrosquamatus

一种新的烙铁头蛇毒肌肉毒素
徐天瑞 王婉 瑜 孟庆雄 黄玉辉 吕秋敏 熊郁良 
中国科学院昆明动物研究所昆明 650223 
摘要用分子筛和快速蛋白质液相色谱FP LC从烙铁头Tr imeresur us m ucrosquam atu s蛇毒中分离了一个新的碱性肌肉
毒素命名为 TM P B 它的分子量为 16 000 等电点为 92用蛋白质序列仪测定了其 N端24 个氨基酸残基T M PB 与其
他两个从同种蛇毒中分离到的碱性磷酯酶 A2的同源性分别为 417和542 TM P B 没有明显的磷酯酶 A2水 解活性 但
它的肌肉毒性和血小板聚集抑制活性却极强其肌肉毒性和血小板聚集抑制活性可被肝素所抑制
关键词肌肉毒磷酯酶 A2血小板烙铁头蛇毒
收稿日期1999021 1 接受日期19990414
联系人Tel 868715194279F ax 868715191823emailx utr 163 net
关于召开第二届纪念曹天钦院士国际蛋白质讨论会的通知
为纪念我国著名生物化学家曹天钦院士逝世五周年曹天钦学术基金中国科学院上海生物化学研究所分子 生物学 国
家重点实验室决定于 2000 年6月11 日 至 6月15 日在上海召开第二届国际蛋白质讨论会会议将邀请国内外有关蛋白质研
究领域的著名专家 20 余人作专题讲演会议将分口头报告及贴报两部分 展示各 实验室的研 究成果 还将对 下世纪的分 子
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龚祖埙
488 
AC T A B IOCH IM ICA et BIO P HYSI CA S IN IC A Vol 31No 5