TAP CHI HOA HOC TAP 49(1), 117-121 THANG 2 NAM 2011
TACH VA BU'6'C DAU NGHIEN cCru cAc TOXIN NGAN CUA Noc B6 CAP HETEROMETRUS LAOTICUS
Hoang Nggc Anh', Vo Do Minh Ho^ng', Nikitin Ilya^ Utkin Yuri^
Viin Khoa hpc Vdt lieu (fng dung, Viin Khoa hpc vd Cdng nghi Viit Nam
" Viin Sinh Hod huv ca, Viin Hdn ldm Khoa hpc Liin bang Nga DinTdaso^n 10-8-2010
Abstract
The scorpion Heterometrus laoticus is wide distributed in the South-West Vietnam. The venom of this scorpion has an analgesic and anti-inflammatory activities. The cmde venom of H. laoticus was separated into five fractions by column chromatography on Sephadex G-50. These fractions have different chemical and biological properties, one them (fraction 4) being toxic to mice, the other (fraction 5) - to crickets. Two of the fractions (fraction 2 and 5) affect the process of blood coagulation, while the cmde venom has no such effect. Several polypeptides with molecular masses in the range from 2000 to 4000 Da were isolated from fraction 5 by combination of gel-filtration on Superdex HR75 column and RP-HPLC. By molecular masses these polypeptides can be assigned to the family of short scorpion toxins. Mass-spectrometry analysis showed that one H. laoticus toxin possesses the molecular mass of 3698.8 Da, which is similar to that of potassium channel toxin alpha-KTx 6.13 (3701 Da) isolated from Malaysian black scorpion H. spinifer. The stmcture and biological activity of the isolated toxins is under study.
I.MODAU
Ngc ddc cua cdc ddng vdt Id ngudn gidu cdc chat CO boat tinh sinh hgc. Cdc peptide frong ngc bd cap CO tdc dung Ien cdc kinh ion khdc nhau [1]. Cdc toxin ngc bd cap tac dyng lln cdc kinh kali la chudi polypeptide chiia tir 31 din 39 gdc axit amin vd dugc dn djnh bang 3 hodc 4 cdu disulfit. Cdc toxin ndy liln kit vdi bl mat ngodi tl bdo cua kinh vd cdn fro sy di qua cua ion K^ Tir cudi nhiing ndm 90 ciia thi ky trudc, cdc nhd hod sinh rat quan tdm din nhiing toxm tdc dyng lln cac kinh K^ [2]. Cdc toxin nay dugc sir dung dl tdch cdc kinh K^ dl nghiln ciiu cdu tnic vd chuc nang chiing. Kinh K* la cac protein mdng t i bdo, chiing cd mat trong cdc tl bdo bj kich thich vd khdng bj kich thich. Nhiing kinh ndy ddng vai frd quan frgng trong nhilu qud trinh khdc nhau cua t i bdo nhu: sy kich thich ciia tl bdo, sy giai phdng chat din truyin thdn kinh, sy tilt ra hdc mdn, su diiu chinh thi tich cua ti bdo.... Ngdy nay ngudi ta tim thdy su liln he mat thiet giiia sy hoat ddng bit thudng cua cdc kinh K* vdi mgt sd benh thin kinh. Dd la mdt si benh liln quan din sy biin dii gel cua kenh K"^ cdn mdt sd benh khac liln quan din sy san xudt ra tu khdng thi cua kinh K^
Nhu vdy nghiln ciiu cdc toxin tdc dung lln cdc kinh K"^ cho phIp thu nghiem san xudt mdt si loai thuoc mdi frong y dugc dk diiu tri cdc benh thin kinh nhu Ashmer, Pakinson ....
Tiip theo nhiing nghiln ciiu v i thdnh phin vd duoc tinh cua ngc bd cap H.laoticus [3] frong bdi
bdo ndy chiing tdi trinh bdy mdt sd nghiln ciiu budc ddu vl cdc toxin ngdn (vdi khdi lugng phdn tu 2000 - 4000 Da) tdch tir ngc bd cap H. laoticus.
2. NGUYEN LIEU VA PHUONG P H A P NGHIEN
cut;
2.1. Nguyen lieu
Ngc thu tir bd cap H. laoticus dugc bdt d vimg An Giang vd nudi tiong phdng thi nghiim ciia Viln Khoa hgc Vdt lieu iing dyng. Thirc an ciia bd cap Id chdu chdu vd dl. Kich thich bang xung diln tai ddt thir 5 hodc dot thii 6 ciia dudi bd cap dl thu ngc. Ngc dugc ldm khd tiong binh hut dm cd chiia CaCl2 khan cho din khi cd khdi lugng khdng ddi, vd sau dd giii d nhilt do -20°C cho din khi dung.
2.2. Cac phuong phap tach va khao sat cac hoat chat cua noc bd cap H. laoticus
2.2.1. Tdch cdc phdn dogn cua npc bd cgp
Cdc phdn doan cd hoat tinh khdc nhau dugc tdch ra tir ngc bd cap bdng phuomg phdp lgc gel frIn mdy FPLC (Bio-Rad, My) d nhilt do phdng frIn cac cdt vdi:
1) Sephadex G-50 (1,5 x 100 cm). Cgt da dugc can bdng trudc frong dim ammonium axetat 20 mM (pH 4,7). Lugng ngc chay mdi lan 200 mg, tdc do nia cgt 0,16 ml/phiit.
TCHH, T.49(l), 2011
2) Superdex HK 75 (1 x 30 cm). Cgt da dugc cdn bdng trudc frong dim axetat amonium 10 mM (pH 6,2). Lugng ngc chay moi lln 15 mg, tic dg rira cdt 1 ml/phiit.
Cdc phdn doan peptide tiip tyc tdch bdng phuomg phdp sdc ky Idng cao dp (RP-HPLC) vdi cgt phdn tich Jupiter Cl 8 (4 x 250 mm, 5pm), tic dg rira cgt Iml/phiit. Cgt sdc ky dugc rira bdng gradient tuyin tinh ciia cac dung djch sau: A la 0,1% TEA trong nudc vd B la 0,1% TFA trong axetonitril. Thdi gian rira cgt tii 5% B din 35% B trong 100 phut.
Sy cd mat cua protein trong cac phdn doan sac ky dugc xac dinh theo mat dO quang tai budc sdng 280 nm.
2.2.2. Khdo sdt tinh chdt cdc phdn dogn tdch ra
Dgc tinh lln dgng vdt ciia cdc phdn do^n tdch ra dugc thir lln chugt nhdt trdng (18-20 g) DDY cd hai gidng bdng cdch tilm ven. Lugng protein tilm vdo mdt chugt Id khodng 0,2 mg. Tdc dung cua cdc phdn doan dugc quan sdt trong 24 gid.
Ddc tinh lln cdn triing cua cdc phdn doan tdch ra dugc thu lln di G. assimilis (0,3 - 0,9 g) [4]. Dung djch protein tir 1 din 6 pl dugc tilm vdo phan bin cua byng dl. Ndng do ciia mdi phdn doan dugc su- dung la 1,5 hay 10 pg/pl. Da sir dyng cdc lilu tiem:
2,5; 5; 10; 15; 20; 25; 30 vd 50 pg/gdi di khdo sdt.
C) mdu ddi chiing, dl dugc tilm nudc cat. Tdc dyng
Tdch vd buac ddu nghien cuu...
cua cdc phan doan dugc quan sat trong 24 gid.
Hoat tinh chong ddng mau ciia cac phan dop tdch ra dugc xdc djnh trong cdc xlt nghiem sau: 1) Xet nghi|m throbin sir dung dk xac djnh tic do chuyin hod phdn tii fibrinogen thanh sgi fibrin trong huyit tuang. 2) Fibrinogenoliz - sir dung d l xdc djnh tic dg chuyin boa tiic thdi ciia fibnogen thanh fibrin. 3) APTT (thdi gian boat hod sy ddng mdu cue bg) sir dung dk xdc djnh thdi gian ddng mdu cue bg, sy kiim sodt qud trinh ddng mdu ben frong. 4) Xlt nghipm Prothrombin- su dyng de xac djnh thdi gian prothrombin, ddnh gid sy bat thudng ciia chiic ndng ddng mdu theo con dudng ngoai sinh. Trong tdt cd xlt nghiim, thdi gian dugc tinh khi mdu dong hoan todn vd mlu ddi chimg la nudc cat.
Khoi lugng phdn tii cua cdc peptid dugc xac djnh trin may khoi phd MALDI MS (Bruker Daltonik, Dire).
2. KET QUA VA T H A O LUAN
2.1. T^ch cac phan doan ciia noc bo cap H. laoticus tren gel Sephadex G 50 va khao sat tinh chat cua chung
Sir dyng cdt vdi gel Sephadex G50 dc phdn tich ngc bd cap H. laoticus cho thdy ngc thd tdch ra lam 5 phan doan vdi khdi lugng phan tii khdc nhau (hmh 1). Khdo sdt tinh chdt ciia cdc phdn doan tach ra cho thdy chiing rat khac nhau.
0.30
0.25
0.20-
0.15-
0.10-
0.05-
0.00-
-0.05
0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 Time [minutes]
Hinh 1: Phdn tich ngc bd cap H. laoticus trin cdt vdi Sephadex G 50
Thii ddc tirih cua ndm phan doan tdch ra lln ddng vdt, cu thi Id lln chugt, cho thay cd mdt phdn doan cd ddc tinh dd la phdn doan 4. Sau khi tiem phdn doan 4, chugt bj lilt vd chit sau Igid 40 phiit.
Trong khi dd cdc phan doan khdc khdng cd ddc tinh lln chugt. Tiep theo chiing tdi da khdo sdt dgc tinh ciia cac phan doan tdch ra len cdn tiung cu thi la lln
dl. Kit qua cho thay phdn doan 5 cd tac dung doc ddi vdi dl, trong khi dd phan doan 4 khdng dgc voi dl ngay cd lilu din 10 pg/gde. Qua khao sat cho thdy phdn doan 5 chira cac peptid ndm frong viing 2000 - 4000 Da. Nhu vdy khac vdi ngc bo cap H. laoticus phdn bd d Thai lan, ngc bd cap H. laoticus Viet Nam chiia cdc toxin dgc vdi con
TCHH, T.49(l), 2011 Hodng Ngpc Anh vd cdng su triing ndm frong vung cd khdi lugng phdn tu nhd.
Cdc toxin nay cd khdi lugng phdn tir ndm trong viing 2000 - 4000 Da, frong khi dd toxin HS-1 ddc vdi cdn trung tdch tir ngc bd cap Thdi Lan cd khii lugng phan tu 8293,496 Da [5]. Nghiln ciiu nay cho thiy thanh phdn, tinh chdt cua ngc bd cap khdng chi phu thudc vdo gidng ma cdn phy thudc vdo viing phdn bd
ciia chiing, dieu ndy da dugc quan sat trong ngc rdn [6].
Tilp theo, chiing tdi tiln hdnh nghiln ciiu tdc dung cua cdc phdn doan tdch ra len qud frinh ddng mdu vdi bdn xlt nghiem khdc nhau. Kit qud cdc xlt nghiem nay dugc trinh bay trong bdng 1.
Bdng 1: Tdc dung cua cdc phdn doan lln qud frinh ddng mau vdi nong do 10 mg/ml
^ ^ - - ^ ^ Phan doan
^^^^---..^^ (thdi gian mdu ddng) Cac xet nghiem ^ " ^ ^ ^ ^ (thdi gian mdu ddng d mdu ddi chirngj^-^..^^^
Xlt nghiim throbin ( 10,2 giay) Fibrinogenoliz (12,6 gidy)
APTT (thdi gian boat hod sy ddng mdu cue bg) (8,4 gidy)
Xet nghiim Prothrombin (17,5 gidy)
1, sec
10,6 8,9 6,1 17,8
2, sec
11,6 11,5 5,8
**
3, sec
*
*
*
*
4, sec
9,0 7,7
**
17,9
5, sec
8,8 8,9 215,1
15,1
*: mau dong rat nhanh; **: mau khong dong trong thdi gian quan sat (ban 30 phut).
Qua cdc xlt nghiem (bdng 1) cho thdy, frong xlt nghiem Prothrombin phan doan 2 gdy ra sy khdng dong mdu frong thdi gian quan sat (hom 30 gidy) do tdc dung lln Prothrombin. Ciing tuomg ty nhu vdy phdn doan 4 vd 5 gay ra qua trinh chdm ddng mdu frong xlt nghiim APTT do tdng thdi gian ddng mdu
cyc bd. Dl xdc djnh chinh xdc tdc dyng cua phdn doan 2 frong xlt nghiim Prothrobin vd tdc dyng cua cdc phdn doan 4 va 5 frong xlt nghiim APTT, chung tdi da pha loang cdc phdn doan nay ra gdp ddi vd tiln hdnh lai hai xlt nghiem frIn. Kit qud dugc trinh bdy tiong bdng 2.
Bdng 2: Tdc dyng cua cdc phdn doan lln qud trinh ddng mdu vdi ndng do 5 mg/ml
—~--^___^ Phan doan
^ — ^ , _ _ ^ ^ (thdi gian mdu ddng) Cac xet nghiem ^ ^ ^ ^ ^ ^
(thdi gian mdu ddng d mdu ddi chiing) -^__^^
APTT (thdi gian boat hod sy ddng mdu eye bd) (11,2 gidy) Xlt nghiem Prothrombin (15,2 gidy)
2, sec
*
33,6
4, sec
16,8
*
5, sec
117,8
*
* Cac phan doan khong lam xet nghiem.
Trong xlt nghiim Prothrobin phdn doan 2 klo dai thdi gian ddng mdu hom gdp ddi so vdi mdu ddi chiing (xem bang 2). C) xlt nghiim APTT phdn doan 4 klo dai mdt it thdi gian ddng mdu, frong khi dd phdn doan 5 ldm tdng thdi gian ddng mdu hom 10 lan so vdi mlu dii chiing (xem bdng 2). Nhiing nghiln ciiu ndy cho thay frong ngc bd cap cd hai nhdm toxin gay tac dung chdm ddng mdu vdi hai ca chi khdc nhau.
Khac vdi tdc dung cua cdc phan doan 2, 4 vd 5 lln qua trinh ddng mdu, ngc bd cap thd vdi ndng do 8,3 mg/ml khdng cd tac dung lln qud trinh ddng mdu d bin xlt nghiem frIn. Nhiing khao sdt ndy cho thdy
frong ngc bd cap thd cdc thdnh phdn tuomg tdc vdi nhau vd iic chl mdt sd tinh chdt ciia nhau.
3.2. Tach cac toxin trong phan doan 5 bang phuong phap sac ky FPLC tren cgt vdi Superdex HR 75 va HPLC vdi cgt RP C18 va khao sat tinh chat cua chung
Dl nghiln cuu cac toxin tdc dyng vdi cdn tning chiing tdi da tiln hdnh sdc ky phan doan 5 frIn cdt lgc gel Superdex HR 75. Kit qud phdn doan 5 tdch ra lam 3 phdn doan thii d p sau: 5.1, 5.2 vd 5.3. Cdc phdn doan nay ddng khd sau dd hod tan 1/3 mdi
TCHH, T.49(l), 201 Tdch vd buac ddu nghien cm...
phan doan trong 200 pl nudc cat vd khdo sdt tdc dung cua chiing trong xet nghi|m APTT.
3.2.1. Khdo sdt tdc dung cua cdc peptid trong cdc phdn dogn thic cdp 5.1, 5.2 vd 5.3 ten thai gian ddng
mdu cue bd (APTT)
Kit qud xlt nghiem APTT ciia cac phan do^n thir cap ndy dugc trinh bdy trong 2 bdng sau.
Bdng 3: Tdc dyng ciia cac phan doan thir cap trong xlt nghi|m APTT
Thdi gian mau ddng d mdu ddi chimg (giay) Cac phdn doan
Thdi gian mdu ddng d mdu thir (giay)
18.2 5.1
*
5.2 18,1
5.3 182,0
"mau khong dong trong thdi gian quan sat (han 30 phiit).
Bdng 4: Tdc dyng cua cdc phdn doan thir cap trong xlt nghiim APTT sau khi pha loang ra 5 Idn Thdi gian mdu ddng d
mdu ddi chiing (gidy) Cdc phdn doan
Thdi gian mdu ddng d mdu thir (gidy)
20,9 5.1 164,1
5.2 25,8
5.3 44,2
Qua nhung khdo sdt nay cho thay cdc phdn doan thii cap 5.1 vd 5.3 cd tdc dyng klo ddi thdi gian ddng mdu ddng kl so vdi mdu ddi chiing. Tilp theo chiing tdi phdn tich phdn doan thii cap 5.3 bdng phuang phdp sdc ky Idng cao dp trin cdt C18. Phdn doan thii cdp 5.3 tdch ra lam 15 phdn doan thii cdp khdc. Thir tdc dyng cua cdc phdn doan thir cdp mdi tdch ra lln qud trinh ddng mdu d xlt nghiim APTT cho thdy cdc phdn doan thii cdp 5.3.1 vd 5.3.9 cd tdc dyng klo ddi thdi gian ddng mdu ddng ki Id 67,2 gidy vd 70,0 gidy so vdi mdu ddi chiing Id 15,2 gidy. Dd Id cdc phdn doan chira cdc toxin tdc dyng lln qud trinh ddng mdu. Tir cdc phdn doan nay chiing tdi se tdch, khdo sdt cac toxin vd se cd cdc cdng bd tilp theo.
3.2.2. Tdch vd khdo sdt tinh chdt cua peptid trong phdn dogn thu cdp 5.2
Qua sdc ky Idng cao dp trin cdt C18, phdn doan thii cap 5.2 tdch ra 7 peptid (xem hinh 2A) trong dd da xdc dinh khdi lugng phdn tii cua mdt peptid 5.2.5 la 3698,8 Da (xem hinh 2B). Peptid nay cd khii lugng phdn tir gan giing vdi khii lugng phdn tii cua toxin alpha-KTx 6.13 dugc tdch tii ngc bd cap den H spinifer ciia Malaysia.Toxin nay cd khii lugng phan tii 3701 Da va tac dyng len kenh kali [7]. Nhu vdy peptid 5.2.5 cd thi la toxin ndm trong nhdm cdc
toxin ngdn tdc dung lln kenh K^ Trong tai lieu cho thay tir ngc bd cap H. spinifi ngudi ta da tdch ra mpt loat cdc toxin ngdn, cd tac dyng Ien cac kenh K^ [7].
Cdc peptid tdch tir phan doan 5 ciia ngc bd cap H.
laoticus cd khoi lugng phan tir nlm trong viing 2000 - 4000 Da, tuomg duong vdi khdi lugng phan tu ciia cdc toxin tdc dung len cdc kinh K^
Ngodi phan doan 5.2.5, chiing tdi cdn tdch mot loat peptid khdc tir cdc phan doan thii cap 5.1 va 5.3.. Trong nhtrng nghiln ciiu tiep theo chiing toi se khdo sdt tdc dyng hod sinh cua cac peptid da tdch ra.
Nhu vdy qua nghien ciru so bg chiing tdi nhan thay trong phdn doan 5 cd hai loai toxin dd Id cac toxin tdc dyng len mach mdu vd mdt sd khdc cd khii lugng phdn tir tuomg duang vdi khdi lugng phan tir ciia cdc toxin tdc dyng lln cdc kenh K* Chung toi se khao sdt tinh chat ciia cdc peptit nay vd cdng bi trong nhirng nghien cuu tilp theo.
4. KET LUAN
1. Ngc bd cap H. laoticus dugc tdch ra lam 5 phdn doan bang phuang phdp sac ky cdt vdi Sephadex G50. Trong cdc phdn doan dd cd mdt phan doan cd ddc tinh vdi ddng vat, phdn doan khdc co ddc tinh vdi cdn trimg v a trong dd cd hai phdn doan cd tdc dung vdi qua trinh ddng mdu.
2. Tir phdn doan ddc vdi cdn triing da tach dugc peptid cd khdi lugng phan tu 3698,8 Da. Peptid nay cd khdi lugng phdn tir gan gidng vdi khdi lugng phdn tu cua toxin tdc dung vdi kinh kali la toxin alpha-KTx 6.13 la 3701 Da. Tinh chdt ciia cdc peptid tdch ra sl dugc xdc djnh frong nhirng nghiln ciiu tilp theo.
TAI LIEU THAM KHAO
1. L. D. Possani, B. Becerril, M. Delepierre, J. Tytgat.
Eur. J. Bioehem., V. 264, 287 300 (1999).
2. M. L. Garcia, M. Hanner, H. G. Knaus, R. Koch, W.
Schmalhofer, R. S. Slaughter, G. J. Kaczorowski.
Adv. Pharmacol, Vol. 39, 425 - 471 (1997).
3. Hoang Nggc Anh, Pham Nguyen Dong Yen, Nguyen Thj Mai Huang, Vo phiing Nguyen. Tap chi Hoa hoc, T.47(2),Tr. 133-137(2009).
4. V. G. Starkov, A. V Osipov, Y. N. Utkin. Toxicon, V. 49, P. 995-1001 (2007).
5. N. Uawonggul, S. Thammasirirak, A. Chaveerach, T.
Arkaravichien, W/ Bunyafratchata, W/
Ruangjirachupom, P. Jearranaiprepame, T.
Nakamura, M. Matsuda, M. Kobayashi, S. Hattori, S.
Daduang. Toxicon, V 49, 19 - 29 (2007).
6. T. H. Tsai, H. Y. Tsai, A. Saha, A. Gomes. FEBS Joumal, V. 274, 512 - 525 (2007).
7. Y. Sugahara, S. Nirthanan, K. Kobayashi, T. Kohno, J. Tytgat, P. Gopalakrishnakone, K. Sato. The
TCHH, T.49(l), 2011
proceedings of the 40"^ Symposium on Japanese
Hodng Ngpc Anh vd cdng sir
peptide symposium, P. 283 - 284 (2004).
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B Hinh 3: Sac ky vd khdi pho ciia peptid 5.2.5
(A) sac ky long cao dp ciia phdn doan thir cap 5.2; (B) khdi phd ciia peptid 5.2.5 Lien he: Hoang Nggc Anh
Vien Khoa bgc Vat lieu Ung dung So 1 Mac Dmh Chi
Quan 1, Thanh phi Hd Chi Minh
Email:hnanh(®vast-hcin.ac.vn, Tel. 0945875569
