Solid-pha se Syn thesis and B ioactiv ity Ana lysis of A1Y -HW TX-g : a M utan t of Huwen tox in-g 3

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1 ISSN CN gQ 16 3 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, g A1Y -HW TX-g ) (, (, ) (Fm oc) (Y) 2g ( ) (A 1) A 1Y2 Edm an, A 1Y2, Y A 1 ; A 1, Y 2g,,, Q 516,Q 50 Solid-pha se Syn thesis and B ioactiv ity Ana lysis of A1Y -HW TX-g : a M utan t of Huwen tox in-g 3 W AN G X ian2chun, L IAN G Song2p ing 3 3 (Colleg e of L if e S cience, H unan N orm al U niversity, Chang sha , Ch ina) LUO Ze2m in (D ep artm ent of B iolog ical T echnology, H unan A g ricu ltu ral U niversity, Chang sha , Ch ina) Abstract T he so lid2phase chem ical syn thesis of A 1Y2, a m u tan t of neu ro tox ic pep tide huw en tox in2g ( ), in w h ich T yr w as sub stitu ted fo r the residue A lal of, w as carried ou t u sing the Fm oc chem istry in the au tom atic w o rk ing station fo r p ro tein chem istry. T he syn thetic m u tan t w as iden tified by Edm an degradation and ES I (electro sp ray ion ization ) 2 m ass spectrom etry. T he b io logical activity analysis dem on strated that the syn thetic A 1Y2HW TX2 g, after ox idatively fo lding in glu tath ione con tain ing buffer, exh ib ited the b io logical activity i2 den tical w ith that of the natu ral, indicating that the sub stitu tion of T yr (Y) fo r the first am ino acid A la (A 1) of had no t resu lted in great changes in the active site and the w ho le confo rm ation of and A 1 w as no t related to the b io logical activity of. T he experim en t also dem on strated that the in troduction of Y in to w ou ld con tribu te to the researches of the m echan ism of. Key words H uw en tox in2g, M u tan t, So lid2phase pep tide syn thesis, Sp ider (S elenocosm ia huw ena) Cys17) 225 (Cys92Cys22) 326 (Cys62Cys29) [ 2, 1992, ], [ 1 ] 90, ( ) 2g (huw en tox in2g, ) 3 3 T el: (0731) , Fax: (0731) ,N A la, C L eu. 6 Cys 124 (Cys22 E2m ail: liangsp@public. cs. hn. cn,, ,, : , :

2 [ 3 ] : Fm oc ( N Α 2Fm oc2aa 2 OH ) 5 ;,, TBTU HOB t NMM ; [ 4 ], DM F; C N, 2. Fm oc 20% [ 5, ] Fm oc, DM F 2D 2NM R, A lal (20 25 ), 215 h ( ) : T FA [ 3 ] = , T yr A lal A 1Y2, A lal,, A 1Y2 : A 1Y2, [ 7 ] : 25 m g 215 m l (Fm oc) M illipo re (715 mm 250 mm ) W aters M illen2 ; : Ser n ium 2010, 996 T yr T h r (tbu) ; Glu A sp 011% (O tbu) ; Cys H is A sn (T rt) ; A rg 2, 2, 4, 6, (Pbf) ; L ys (Boc). PEG2PS HC l 37% ) 115 m lgm in R. L au rsen ; A 1Y2 (DM F ) (D T T ) [ 8 ] : (GSH ) (GSSG) A 1Y2 250 m l (TBTU ) 12 (HOB t) N 2 (NMM ) ( T FA ) m o lgl GSH m o lgl GSSG, ph 9. 5) (p iperidine) Sigm a (20 25 ) 24 h, A 1Y2 : PEG2PS HC l W aters 650E : (A ) [ 6 ] : 0105 mm o l PEG2PS HC l, [6 ],,, (011 m o lgl T ris2hc l, 0. 1 m o lgl D T T, m o lgl ED TA, 6. 0 m o lgl, ph 816) (20 25 ) 2 h, ph 3 4, T FA 280 nm, (20 m in, 35% ( 011 m o lgl, m o lgl ED TA, ph W aters P21 (10 mm 100 mm ) 0. 2 m o lgl N ah 2PO 4; (B ) 0. 2 m o lgl N a2h PO 4; (C), HOB t m o lgl N ac l; (D ), - OH ( H PL C ) N ac l (42 ) [ 40 m in 0% 80% (C) ] ; ph, A 1Y2 C (N a2h PO 4) 110 m lgm in (L eu, N Α 2Fm oc2l 2L eu2opfp ) H 1Y2 : A 1Y2 - OH M icrom ass L CT ; N

3 3 : 2g A 1Y2 359 [ 9 ] M illigen 6600, 280 nm ; [10 ] : [ ], 2 A 1Y2 : 2, C 18 A 1Y2 ( ) ; (N ac l ) F ig % O 2 5% CO 2 ; H z (, ),, A 1Y2HW TX2g 2 A 1Y A1Y -HW TX-g, m g A 1Y2 H PL C F ig A1Y -HW TX-g A 1Y2 24 h, F ig. 2 Ion2exchange H PL C p rofile of the renatured A 1Y2 E luted by a N ac l gradient, 0% 70% (C) over 30 m in F ig. 1 A nalytical ch rom atogram of crude synthetic A 1Y2 by RP2H PLC Co lum n: YW G C18 10 m reverse2phase co lum n, 3. 9 mm 300 mm. Buffer: (A ) 0. 1% TFA in w ater; (B) 0. 1% TFA in ace2 tonitrile. Gradient: 0% 93% (B) in 35 m in. F low rate: 0. 7 m lgm in. T emperature: 40. D etection: 280 nm, F ig. 2, ph (N a2h PO 4) F ig. 3. A 1Y2 H PL C 213 A1Y -HW TX-g ( F ig. 4 ), A 1Y2 (M + H ) , ( ), A 1Y2 6 A 1Y2 N 7,, A 1Y2 N H 2N 2YCK2 GV FD -, N H 2N 2 A CKGV FD -,,A 1Y2 N F ig. 1,,

4 360 16, F ig. 5. F ig. 5 B lockades of neurom uscular transm ission by A 1Y2 and natural a: Responses of m uscle elicited by direct stim ulation after blockade F ig. 3 T he second ion2exchange H PL C p rofile of the rena2 tured A 1Y2 E luted by a ph (N a2h PO 4) gradient, 0% 100% (B ) over 65 m in, Y A A 1Y2,,, ( ) : A la 1110 (1), A rg 0195 (1), A sx 3135 (4), Glx 1104 (1), Gly 1190 (2), H is 0192 (1), L eu 1128 (1), L ys 5176 (6), Phe 0188 (1), P ro (2), Ser 0187 (1), T h r 0181 (1), T yr 0170 (1), V al (2). T rp Cys, HOB t, PEG2PS F ig. 4 N anoelectro sp ray analysis of the renatured A 1Y2 F ig. 5, ( 512 m o lgl ), A 1Y m in ( ) 2, A 1Y2, F ig. 5, HC l ( 215 h) 97%,,,,,, [ 12 95% ( ] ) A 1Y2,,,,, A 1Y2, 214, A 1Y2, A 1Y2, ; A 1Y2 2

5 3 : 2g A 1Y2 361, H is,, 4 (F ig. 6a) [ 13 ] ; ( ) ( ) T yr GSH 2GSSG Cys2(Cys) 2, A lal,,, 2 h [ 14, 15 ], (F ig.,, (20 25 ) 6b), 125 I2A 1Y2 A 1Y2 GSH 2GSSG 24 h,, A 1Y2,,,,,, [ 16 ], GSH 2GSSG [ 17, ] A 1Y2,,,,, H PL C T yr A lal, T yr A lal ; A lal,, F ig. 6 L abeling of natural and A 1Y2 [ 18 ] Fm oc 28 N A lal C 4, A lal M icrom ass,! A 1Y2, A lal (References) 125 T yr, I In F ig. 6a, the CPM (the counts per m inuts) of 125 I labeled2 CPM co llected. (peak 1) accounts fo r less than 10% of to tal In contrast, the CPM of 125 I labeled2a 1Y2 (peak 1) accounts fo r mo re than 30% of to tal CPM co llected in F ig. 6b, 1,, (W ang J ia2fu, Peng X ian2jin, X ie L i2p ing. A new species of the genus Selenoco sm ia from south China. A c2 ta S ci N atu r U niv N orm, H unan), 1993, 16: 51 54

6 Zhang D Y, L iang S P. A ssignm ent of the th ree disulfide bridges of H uw entoxin2g, a neuro toxin from the sp ider Seleno2 co sm ia huw ena. J P rotein Chem, 1993, 12 (6) : Q u You2xing, L iang Song2p ing, D ing J i2zhen, L iu X iu2chai, Zhang Ren2ji, Gu X iao2cheng. P ro ton nuclear m agnetic reso2 nance studies on huw entoxin2g from the venom of the sp ider Selenoco sm ia H uw ena: 2, Three2dim ensional structure in so lu2 tion. J P rotein Chem, 1997, 16 (6) : (L iang Song2 p ing. H uw entoxin and analogous pep tides. Chem L if e), 1997, 17 (5) : ,, : 2g ( ) (Chen X iang2ding, Peng Kuan, L iang Song2p ing. The effect of H uw entoxin2g on the iso lated guinea p ig ileum. L if e S ci R es), 1998, 2 (4) : A therton E, Sheppard R C. S olid P hase P ep tid e S y nthesis: A P ractical A pp roach. O xifo rd: O xifo rd U niversity P ress, 1989: Saxena V P, W etlaufer D B. Fo rm ation of th ree2dim ensional structure in p ro teins, a rap id nonenzym ic reactivation of re2 duced lysozym e. B iochem istry, 1970, 9: ,, 2g K3A 2 (W ang X ian2chun, L iang Song2p ing, L uo Ze2m in. Chem ical synthesis and physio logical activity analysis of K3A 2 : a m utant of huw entoxin2g. L if e S ci R es), 1998, 2 (2) : N ish io H, Kum agaye K Y, Chen Y N. Synthesis and disulfide structure determ ination of Ξ2agatoxin IVA. P ep tides, Chem 2 istry, S tructure and B iology. P roceed ings of the T hirteenth A m erican P ep tide Sy np osium. L eiden: ESCOM p ress, 1993: L iang S P, Zhang D Y, Pan X. P roperties and am ino acid se2 quence of huw entoxin2g, a neuro toxin purified from the venom of the bird sp ider Selenoco sm ia huw ena. T ox icon, 1993, 31 (8) : Zhou Pei2ai, X ie X ian2jin, L im an, Yang Dong2m ei, X ie Zuo2 p ing, Zong X iang, L iang Song2p ing. B lockade of neurom uscular transm ission by H uw entoxin2g from the venom of the chinese bird sp ider Selenoco sm ia huw ena. T ox icon, 1997, 35: Sarin V K, Kents B H, T am T P. Q uantitative monito ring of so lid2phase pep tide Synthesis by the ninhydrin reaction. B iochem, 1981, 34: A nal 13 N adasdi L, Yam ash iro D, Chung D, Katalin T arczy2ho rnoch, A driaenssens P, Ram achandrran J. Structure2activity analysis of a conus pep tide blocker of N 2type neuronal calcium channels. B iochem istry, 1995, 34: L ozanov V, Guarnaccia C, Patthy A, Fo tt S, Pongo r S. Syn2 thesis and cystinegcysteine2catalyzed oxidative fo lding of the A 2 m aranth Α2am ylase inh ibito r. J P ep tid e R es, 1997, 50: D rakopoulou E, Soph ie Zinn2Justin, Guenneugues M, Gilquin B, M enez A, V ita C. Changing the structural context of a functional Β2hairp in. J B iol Chem, 1996, 271 ( 20) : P ierret B, V irelizier H, V ita C. Synthesis of a m etal binding p ro tein designed on the ΑgΒ scaffo ld of charybdo toxin. P ep tid e P rotein R es, 1995, 46: Int J 17 C reigh ton T E. D isulfide bond fo rm ation in p ro teins. M ethod s E nzym ol, 1984, 107: ,,,, 2g ( ) 28 (L iao Q ian2ning, L uo J ing2chu, Zhou Pei2 ai, Gu X iao2cheng, L iang Song2p ing. So lid2phase chem ical syn2 thesis and the bioactivity identification of the 28 residue ana2 logue of H uw entoxin2g. Ch in J B iochem M ol B iol), 1999, 15 (5) :