Pur if ica tion and Partia l Character iza tion of Hem orrhag ic Tox in from the Venom of D ienagk istrodon acu tus

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1 V o l 15,N o 5 Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy O ct, 1999 DaHT- (, 65022) (, ), Sephadex G275, D EA E2Sephadex A 250, Sephadex G2200 PBE, (D ienag k istrod on acu tus) (D ah T 2),, 487 SD S2PA GE,, p I Λg,, 168, A 2 D ah T 2 I, 118% Α 5611% Β 1211% ; ED TA, Α Β 11% 2614% 4612% 1615%,,,,, Pur if ica tion and Partia l Character iza tion of Hem orrhag ic Tox in from the Venom of D ienagk istrodon acu tus YE W en juan, RAN Yonglu,LA I R en (K unm ing Institute of Z oology, the Chinese A cademy of S ciences, K unm ing 65022) GON G Guang lin, YU Fan, ZEN G Zhaoquan (E xp erim ent Center, Y unnan U niversity, K unm ing ) Abstract A new hem o rrhagic tox in, nam ed D ah T 2, w as purified from the venom of D ienag k2 istrod on acu tus u sing five step s of Sephadex G275 filtration, D EA E2Sephadex A 250 ch rom atogra2 phy, Sephadex G2200 filtration and tw o Po ly2buffer2exchanger ch rom atofocu singṡ T he purified sam p le w as hom ogeneou s as judged by SD S2po lyacrylam ide gel electropho resiṡ D ah T 2 had m o lecu lar w eigh t of estim ated by SD S2PA GE, w h ile it w as u sing determ ination of am ino acid au toanalyzer w ith 487 am ino acid residueṡ W hen the purified sam p le w as judged on the gel of isoelectric focu sing electropho resis, it had isoelctric po in t (p l) of 5 50 T he m in im um hem o rrhagic do se w as m easu red to be 216Λg, w hen D ath 2 w as in jected in to m ice T he m in i2 m um hem o rrhagic do se (M HD ) w as defined as the low est do se w h ich cau sed an area of hem o r2 rhage of 5mm in diam eter 6 hou rs after subcu taneou s in jection T he purified sam p le w as assayed by u sing casein as sub strate, w h ich exh ib ited an active un it of 168 N o argin ine esterase and pho spho lipase A 2 activity w ere ob served w ith TAM E, BA EE and lecith in as sub strates, respec2 tively W hen ab so rben t bands of am ide I of D ah T 2 w as estim ated by Fou rier2tran sfo rm 2infrared (95B09M ) Tel: (0871) , Fax: (0871) , E2m ail: yangdr@m ail kiz ac cn,, , : , : China Academic Journal Electronic Publishing House All rights reserved

2 5 : D ah T 2 76 (FT 2IR ) spectrom eter, its con ten ts of secondary structu re in aqueou s so lu tion w ere Α2helix of 118%, Β2p leat sheet of 5611%, and tu rn of 1211%, respectively1t here w as no uno rdered structu re in the sam p le A fter the addition of ED TA, w h ich m oved m etal ion s, the Α2helix, Β2 p leat sheet, tu rn, and uno rdered structu re w ere 11%, 2614%, 4612%, and 1615%, respectively T heir hem o rrhagic and p ro teo lytic activities at the sam e tim e w ere lo sṫ It cou ld be con sidered that m etal ion s, particu larly Zn ion, p layed an im po rtan t ro le in m ain tain ing secondary structu re of the hem o rrhagic tox in s purified from snake venom ṡ It w as very clear that the purified sam p le w as a m etallop ro teinase T he yield of D ath 2 w as 0 5% on ly Key words D ienag k istrod on acu tus venom, H em o rrhagic tox in, Physical and chem ical p roperties, Fou rier tran sfo rm infrared spectrum, PBE 212, 5 (22 2 g), 5,, 0 1 m l ( ), 6 h,,,,,, [ 1 6 ],, (M HD ) 5 mm, 21,, SD S2PA GE : T = 10%, C= 217%, 1% SD S; : T = 4%, C = 217% ; 20 ma, 4 h,, 01125% R % 10% 1 1 h, 5% 7% (D ienag k istrod on acu tus) L KB ( IEF ) H ITA CH I B io2r ad FT S Sephadex G275 Sephadex G D EA E2Sephadex A 250 Po ly2buffer2exchanger (PBE ) Pharm acia Am pho line (ph ) L KB T EM ED Β2 F luka BA EE TAM E Sigm a A n thony T T u [ 7 ] [ 8 ] A 2 B io2r ad FT S, H gggdgt e GegKB r 4 cm - 1, B io2r ad 0 01 cm - 1 :, A nchun [ 11 ],, Sephadex G275,,, 16, D EA E2Sephadex A 250,, Sep hadex 9 G2200 ;, Sephadex G2200, (HW HH ) 20 PBE,, cm - 1, K China Academic Journal Electronic Publishing House All rights reserved

3 m l, 4 g1 h, 280 nm 500 m g 8 m l 0101 m o lgl N H 4A c (ph 516), 1, 6 m l 0101 m o lgl N H 4A c, 0101 m o lgl N H 4A c F ig 1 Sephadex G275 gel filtration Sephadex G275 ( cm ),, (F ig 1) 1 2 (ph 516), 0101 m o lgl D EA E2Sephadex A 250 ( cm ),,, m o lgl N ac l ( m l N H 4A c ), 4 m l, 1 g15 m in, 280 nm, (F ig12) F ig , Sephadex G2200 ( cm ), ( ), Sephadex G2200 PBE ( cm ), m o lgl N H 4A c, ph (ph 414 F ig 2 D EA E2Sephadex A 250 ch rom atography F ig Po lybufferexchanger ch rom atofocusing China Academic Journal Electronic Publishing House All rights reserved

4 5 : D ah T ) (F ig1a) F ig a 2 2 Table 1 Am ino acid compo sition of hemo rrhagic toxin, PBE,, (F ig b), SD S2PA GE (F ig 4) 015% 12 5,, 011 m l, 1186 Λg ;, 172 Λg;, 5158 Λg;, 7144 Λg;, 91 Λg, ( ), Am ino acids Resides Am ino acids Residues A sp 55 M et 24 T h r 21 Il 18 Ser 42 L eu 24 Glu 70 T yr 8 P ro 5 Phe 4 Gly 4 L ys 5 A la 16 H is 12 Cys n A rg 12 V al 47 T rp n To tal am ino acid residues 487 Calculated mo lecular w eight M o lecular w eigh t on SD S2PA GE n: no determ ination (M HD ) 216 Λg, A m o lg SD S2PA GE (F ig 4), L ED TA,,,,, 6 h,, 0102 m o lgl ED TA , p I 5150, 0102 m o lgl, 487 ED TA (T ab le 1), 54 57, A sp Glu L ys A rg, 15 A nchun [ 11 ], Su rew icz [ 12 ] Bandekar [ 1 ] F ig 5, F ig 6 T ab le 2 F ig 4 SD S2po lyacrylam ide gel electropho resis A Purified samp le; B Standard mo lecular w eigh t p ro teins: 1 Pho spho rylase b ( ) ; 2 Bovine Serum A lbum in (67 000) ; O valbum in ( 4 000); 4 Carbonic A nhydrase (0 000) ; 5 Soybean tryp sin inh ibito r (20 100) ; 6 Α2L actal2 bum in (14 400) %, = gm in 1000gm g, 1681, TAM E BA EE F ig15 FT 2infrared spectro scopy of D ah T 2at the am ide I re2 gion of D 2O so lution A : Secondary2derivative spectra; B: D econvo lute am ide I spec2 tra; C: P rim ary FT 2infrared spectra China Academic Journal Electronic Publishing House All rights reserved

5 F ig 6 FT 2infrared spectrogram of D ah T 2 at the am ide I re2 gion of D 2O so lution after adding 0 02 mo lgl ED TA treat2 m ent and dialysisa : Secondary2derivative spectra; B: D econvo2 luted am ide I spectra; C: P rim ary FT 2infrared spectra Table 2 Effect of m etal ions on the so lution confo rm ations of hemo rrhagic toxin (D ah T 2) from the venom of D ien2 ag k istrod on acu tus Α2H elix Β2Sheet Turn U no rdered co il N o ED TA 118% 5611% 1211% - A dded ED TA 1110% 2614% 4612% 1615% 4, [ 4 ],, , Zhong L Zhu [ 5 ] 1, , p I, 510, N ikai [ 9 ] M o ri [ 10 ] 5 5,, ,,, Yong, L R an [ 2 ] Sanchez [ 7 ] (L achesis, m u ta ) M o ri [ 1 ] (C rota lus ruber ruber) [ 6 ] 2 (D ah T 21 D ah T 22), 2 500, , D ah T 2, , 5150, 216 Λg, ED TA, D ah T 2,, F ig cm - 1 Α2,, cm cm cm cm - 1 Β2,, (F ig 6), cm - 1 Α2, 1 62 cm cm - 1 Β2,, cm cm cm - 1, 1 64 cm - 1 [ 14 ] CD ED TA D ath 21 D ath 22,,, References 1 M o ri N, N ikai T, Sugihara H, T u A T B iochem ical characteriza2 tion of hemo rrhagic toxins w ith fibrinogenase activity iso lated China Academic Journal Electronic Publishing House All rights reserved

6 5 : D ah T from C rotalus ruber ruber venom A rch B iochem B iop hy s, 1987, 25: Ran Y L, Zheng S D, T u A T B iochem ical characterization of two hemo rrhagic p ro teases from the venom of L achesis m uta (Bush2 m aster) Chem R es T ox icol, 1988, 1: 7 42 Sanchez E F,M agalhaes A, D im iz C R, Purification of a hemo r2 rhagic facto r (LH F21) from the venom of the bushm aster snake, L achesis m u ta m u ta T ox icon, 1987, 25: ,, (A g k istrod on acu tus) : p ro teinase) and characterization of hemo rrhagic toxins from the İ (Xu Xun, Xu venom of the hundred2pace snake (A gk istrodon acutus) T ox i2 Zhengyu, W ang Chun Snake venom of A gk istrodon acutus (five2 pace snake ) İ Purification of three toxic components A cta B ioch im B iop hy s S in), 1982, 14 (1) : Zhu Z L, Gong W M, Zhu X Y, T eng M K, N iu L W Purifica2 tion, characterization and confo rm ational analysis of a haemo r2 rhagin from the venom of A gk istrodon acutuṡ T ox icon, 1997, 5 (2) : , 1987, 167: (Tao Yong, Ran Yonglu Purification and biochem 2 ical characterization of two hemo rrhagic toxins from the venom D ienag k istrod on actus (from H unan) Ch in B iochem J ), 1995, 11 (2) : T u A T, D avid S M, P ro tein nature and som e enzym atic p roper2 ties of the lizard H eloerm a susp ectum susp ectum (Gila monster) venom Comp B iochem P hy siol, 1967, 22: , A (Chen Yuanchung,W u Sh inlu A comparison of pho spho lipase A activity from the snake venom s of ten species in Ch ina Z oolog2 ical R esearch), 1982, 2 (4 ) : N ikai T, Ish izak i H, T u A T, Sugihara H, P resence of zinc in2 p ro teo lytic hemo rrhagic toxin iso lated from A gk istrodon acutus venom Comp B iochem P hy siol, 1982, 72C: M o ri N, N ikai T, Sagihara H, Purification of a p ro teinase (A C52 con, 1984, 22: A nchun D, H uang P, Caughey W S P ro tein secondary struc2 tures in w ater from second2derivative am ide I infrared spectra B iochem istry, 1990, 29: Surew iczw K, Szabo A G,M antsch H H Confo rm ational p rop2 erties of azurin in so lution as determ ined from reso lution2en2 hanced Fourier2T ransfo rm infrared spectra E ur J B iochem, 1 Bandekar J Am ide modes and p ro tein confo rm ation B ioch im B iop hy s A cta, 1992, 1120: , (Tao Yong, Ran Yonglu C ircular dichro ism studies on hemo rrhagic toxin 1 and 2 from the venom of D ien2 agk istrodon acutus Ch in B iochem J ), 1995, 11 (2) : China Academic Journal Electronic Publishing House All rights reserved