SO SNH HOT CA MT S ENZYM BO V TN THNG OXY HO V PH BNG ADN CA C BRADYBAENA SIMILARIS THU THP M V CT TIN TNH NG NAI

ng Quang Hng, Nguyn Quang Huy, Phan Tun Ngha Trng i hc Khoa hc T nhin 1. M U Cc tc nhn oxy ho nh peroxit hydro, cc gc t do superoxit (O2.-), gc hydroxyl (OH.) lun c hnh thnh trong cc hot ng sng ca cc c th sinh cng nh trong qu trnh tng tc ca sinh vt vi mi trng. Chnh nhng tc nhn ny l nguyn nhn gy ra cc tn thng oxy ho [5]. chng li tc dng tn thng ca cc tc nhn ny, sinh vt hnh thnh cc c ch bo v khc nhau, trong ch yu da vo cc enzym c hot tnh phn hu cc hp cht oxy ho nh l superoxit dismutase (SOD) trit tiu gc superoxit hay NADH oxidase (NOX), catalase (CAT), alkyl hydroperoxide, glutathion reductase c tc dng phn hu peroxit hydro...Chnh v vy, nghin cu cc enzym ny s l c s tm hiu s p ng ca c th vi cc tc nhn oxy ho khc nhau bao gm cc tc nhn ni sinh cng nh ngoi sinh. Bi bo ny trnh by cc kt qu nghin cu bc u v vic so snh hot ca enzym SOD, CAT, NOX, ph bng protein v ADN ca loi c Bradybaeana similaris thu thp t hai vng M v Ct Tin (tnh ng Nai), t c s cho cc nghin cu tip theo v s tc ng ca mi trng ln c th. 2. NGUYN LIU V PHNG PHP NGHIN CU Nguyn liu. Loi c Bradybaena similaris (Ferussae) c thu thp M v Ct Tin thuc tnh ng Nai, c PGS. TS. Nguyn Xun Qunh nh tn khoa hc. Mu vt c gi sng hoc gi 80oC cho n khi phn tch. Mi dng cho phn tch ph bng RAPD bao gm: OPA4 (5'-AATCGGGCTG), OPA10 (5'-CAGGCCCTTC), OPA12 (5'-GGGCGGTACT) v thang chun ADN c t t hng Invitrogen (M). Xanthine, xanthine oxidase, nitro tetrazolium blue (NTB) v riboflavin c mua t hng Sigma (M), cc ho cht cn li u t tinh sch dng cho phn tch. Phng php nghin cu: Dch chit mu c c chun b bng cch loi b v, ct ly phn c v nghin trong ci s 4oC vi t l 1g nguyn liu: 10 ml m chit (Tris-HCl 100mM, pH 7 cha KCl 50mM). Mu sau khi nghin c ly tm tc 10.000 vng/pht 4oC trong10 pht thu dch trong dng cho phn tch protein v hot enzym.

ADN ca c c tch chit theo phng php c m t trong [14] sau c ho tan tr li trong m TE (Tris-HCl, 10 mM, pH 8 c cha 2 mM EDTA) Protein c xc nh theo phng php ca Lowry [8], dng albumin huyt thanh b lm cht chun. Hot SOD c xc nh theo phng php Mc Cord v Fredovich [11]. in di trn gel polyacrylamit c tin hnh theo phng php ca Laemmli [7]. Ph bng SOD c xc nh theo phng php ca Beauchamp v Fredovich [4]. Hot NOX c xc nh theo phng php Poole & Clairborne [15]. Hot catalase c xc nh theo phng php ca Thibodeau v cng s [17] v Mottola v cng s [12]. Phn ng RAPD-PCR Hn hp phn ng RAPD (25l ) c cha 0,2mM mi loi dNTPs, 1l mi (20 pmol) v 2,5 l10X m phn ng (c 25mM MgCl2) v 2,5 n v Taq ADN polymerase. Giai on bin tnh c thc hin 95oC trong 3 pht; tip theo 45 chu k, mi chu k gm cc bc thay i nhit v thi gian: 94oC-30 giy; 36oC- 45 giy;72oC-1 pht tip theo l 72oC trong 7 pht. Sn phm RAPD c in di trn gel agarose 1%, nhum vi ethidium bromide, soi di n UV v chp nh. 3. KT QU NGHIN CU 1. So snh hot SOD, NOX, CAT ca c Bradybaena similaris thu thp t M (BSM) v Ct Tin (BSCT). 1.1 Hot SOD ca c BSM v BSCT

Hnh 1: Hot SOD tng s ca c BSM v BSCT.

A: Hot tnh theo mg cht kh, B: Hot tnh theo mg protein. Kt qu phn tch hot SOD ca mu c hnh 1 cho thy hot SOD ca c B. similaris M (BSM) l khong 22 v/mg cht kh cn Ct Tin (BSCT) l khong 40 v/mg cht kh v hot ring (HR) tng ng l 1100 v/mg protein v 2600 v/mg protein. Nh vy hot SOD ca BSM l cao hn so vi ca BSCT l 1,8 ln. Kt qu nghin cu ph bng SOD (hnh 2) cho thy hai mu c BSM v BSCT u c ph bng nh nhau vi 1 bng r nt c Rf 0,13 v mt vng sng c th cha vi bng SOD vi Rf nm trong khong 0,23 - 0,25. Nh vy, s khc bit v hot SOD ca c BSM v BSCT trn khng lin quan n s lng SOD ca chng c mt trong mu phn tch.

Hnh 2: Ph bng SOD ca c BSM v BSCT (A: BSM, B: BSCT)

SOD l nhm enzym c cofact l ion kim loi [5,11]. s b tm hiu ion kim loi c mt trong SOD ca c Bradybaena similaris, chng ti tin hnh thm tch dch chit mu trong m c cha EDTA nhm loi b cc ion kim loi ra khi enzym ca mu nghin cu, sau tin hnh xc nh hot SOD vi s b sung cc ion kim loi khc nhau. Kt qu thu c hnh 3 cho thy qu trnh thm tch khng lm mt hon ton hot SOD ca mu c BSM cng nh BSCT. Tuy vy, trong s 4 loi ion kim loi ho tr 2 (Ni2+, Mn2+, Fe2+, Zn2+) c b sung vo hn hp phn ng th ch Fe2+ c kh nng lm tng mt phn hot SOD (khong 5%) ca BSM cng nh ca BSCT. Vic b sung cc ion kim loi khc c tc dng km hm mt phn hot SOD ca BSM v BSCT. C th SOD ca BSM v BSCT u cn Fe2+ cho hot ng xc tc ca SOD v SOD ca chng c th thuc nhm FeSOD.

Hnh 3: nh hng ca ion kim loi ln SOD ca c BSM v BSCT

Kt qu phn tch bn ca SOD trong mu c BSM v BSCT vi nhit (hnh 4) cho thy SOD ca c hai mu nghin cu u khng bn vi nhit. Sau khi x l nhit 80oC trong 15 pht, hot SOD ca c hai mu c BSM v BSCT u b mt hon ton v khng c s sai khc ng k v mc bn vi nhit gia hai mu nghin cu.

Hnh 4: bn vi nhit ca SOD ca c BSM v BSCT

Hnh 5: Hot NOX ca c BSM v BSCT A: Hot tnh mg cht kh, B: Hot tnh theo mg protein.

Kt qu xc nh hot NOX (hnh 5) cho thy hot NOX ca mu c BSM l

0,0022 v/mg cht kh v ca c BSCT l 0,0027 v/mg cht kh. HR NOX tng ng ca BSM v BSCT l 0,12 v/mg protein v 0,175 v/mg protein. Nh vy l hot NOX ca mu c BSCT cao hn BSM khong 1,2 ln. Nghin cu v nh hng ca nhit ln hot NOX (hnh 6) cho thy NOX ca dch chit BSM t ra km bn nhit hn so vi BSCT, c th khi x l nhit 60oC v 80oC dch chit ca c BSM mt tng ng 25% v 98% hot , trong khi dch chit ca BSCT ch mt tng ng 30% v 60% hot .

Hnh 6: bn vi nhit ca NOX ca c BSM v BSCT

1.3. CAT ca c BSM v BSCT Catalase (CAT) l enzym ch yu gia vai tr phn hu cc H2O2 trong hu ht cc c th sinh vt. Kt qu xc nh hot CAT ca mu c hai vng M v Ct Tin (hnh 7) cho thy hot CAT ca mu c BSM l khong 16 v/mg cht kh v ca c BSCT l khong 20v/mg cht kh. Hot ring CAT ca c BSM l 900 v/mg protein v ca c BSCT l 1400 v/mg protein. Nh vy hot CAT ca mu BSCT cao hn ca BSM 1,25 ln.

Hnh 7: Hot CAT ca c BSM v BSCT. A: Hot tnh theo mg cht kh, B: Hot tnh theo mg protein. Kt qu nghin cu nh hng ca nhit ln hot CAT (hnh 8) cho thy khng c s khc nhau ng k v mc nh hng ca nhit ln hot CAT gia mu

BSM v mu BSCT. CAT trong c hai mu u km bn vi nhit, gn nh mt hon ton hot nhit 80oC.

Hnh 8: bn vi nhit ca CAT ca c BSM v BSCT

Hnh 9: Ph bng protein ca mu c BSM v BSCT. (A: BSM, B: BSCT).

Kt qu phn tch ph bng protein ca dch chit mu c bng in di (hnh 9) cho thy c hai loi mu c BSM v BSCT c in di v c bn l ging nhau vi khong 15 bng chnh. Tuy nhin, chng ti cng pht hin thy rng trong ph bng protein ca c BSM (ct A) thy c xut hin thm mt bng protein vi khi lng phn t khong 21kDa v bng protein ny khng c mt c BSCT (ct B). 2. S a hnh di truyn ca c Bradybaena similaris M v Ct Tin. tm hiu s khc bit v ph bng ADN ca mu c BSM v BSCT chng ti s dng k thut RAPD-PCR vi h thng mi ngu nhin. Kt qu phn tch ph bng RAPD vi 3 mi n l OPA4, OPA 10 v OPA 12 cho thy c s ng hnh rt cao gia hai mu BSM v BSCT, c th chng ti khng pht hin ra s sai khc no v cc bng c nhn bn. tip tc nghin cu su hn chng ti tin hnh thc hin phn ng RAPD ci tin bng cch phi hp tng cp mi vi nhau. Kt qu hnh 10 cng cho thy s ng hnh di truyn cao gia mu BSM v BSCT. Tuy nhin trong

trng hp ny chng ti pht hin thy trong s 36 bng nhn bn th mt bng vi kch thc khong 500bp ch thy mu c BSCT (hnh 10, ct 4) v bng c kch thc khong 750bp ch thy mu c BSM (hnh 10, ct 5). C hai bng u do kt hp mi OPA4 vi OPA12.

Hnh 10: Kt qu in di sn phm RADP-PCR ca c BSM v BSCT vi cc cp mi OPA4+OPA10 (ct 2 v 3); OPA4+OPA12 (ct 4 v 5) v OPA10+ OPA12 (ct 6 v 7)

Ct 1: Thang chun ADN 1kb Ct 2,4,6: mu c BSCT; Ct 3,5,7: mu c BSM. 4. THO LUN Superoxide dismutase, NADH oxidase, catalase l cc enzym bo v tn thng oxy ho c mt ph bin trong cc c th sinh vt. Rt nhiu enzym c tinh sch, nghin cu tnh cht, gen m ho cng nh xc nh v mt chc nng t cc i tng sinh vt khc nhau [5]. Tuy vy cho n nay vn cn rt t cng trnh nghin cu v s lin quan ca cc enzym ny vi cc tc ng ca mi trng. Cc kt qu nghin cu ca chng ti v SOD, NOX v CAT c B. similaris, thu thp hai vng M v Ct Tin cho thy hot enzym tng s trong mu Ct Tin lun cao hn so vi hot ca cc enzym tng ng M . C th hot SOD, NOX v CAT ca mu BSCT cao hn so vi ca BSM tng ng l 1,8 ln (hnh 1), 1,2 (hnh 5) v 1,25 ln (hnh 7). Khi phn tch ph bng SOD bng k thut in di cho thy s ging nhau gia cc mu BSM v BSCT (hnh 2). S ging nhau v SOD cn c th hin mc nh hng ca cc ion kim loi ln hot SOD (hnh 3), iu ny chng t s khc nhau v hot SOD gia hai mu l khng lin quan n s SOD c mt trong mu nghin cu. Khc vi SOD, chng ti cha c iu kin phn tch ph bng ca NOX cng nh ca CAT, v vy chng ti cha th khng nh c s sai khc v hot ca NOX v CAT ca cc mu nghin cu gia hai vng thu mu M v Ct Tin c phi l do s khc nhau v s lng enzym c mt hay do mc biu hin ca cc gen hay do cc nguyn nhn khc. Nhng sai khc v hot ca mt s enzym oxy ho nh glutathionine peroxidase, glutathionine reductase chut do x l bng dioxin cng

c cp trong mt vi nghin cu trc y [1,2,6]. Tuy vy cc tc gi cng cha c cc iu tra chi tit v nhng sai khc ny. Mayuree v cng s [9,10] khi nghin cu tc ng ca H2O2 v Fe2+ ln s biu hin ca gen catalase (kat) hay peroxidase (per) B. subtillis cho thy cc hp cht ny khng tc ng trc tip ln cat hay per ca t bo m hot ho mt s protein iu ho m c i lc vi promoter ca cc gen ny, do dn n lm tng hot ca cc enzym. S c mt ca bng protein 21 kDa mu BSM m khng c mu BSCT (hnh 9) l c s ngh rng s khc nhau v hot SOD, NOX v CAT c th lin quan n c ch hot ho/c ch biu hin gen tng t nh cc gen kat v per B. subtillis. Kt qu phn tch s nh hng ca nhit ln hot NOX ca mu c B. similaris cng cho thy c nhng s khc bit nht nh v mc nh hng vic x l nhit ln hot enzym ny gia cc mu BSM v BSCT, mc du khng thy c s khc bit ng k v nh hng ca nhit ln hot SOD v CAT gia hai mu BSM v BSCT. Tm li, chng ti pht hin c nhng sai khc v hot SOD, NOX, CAT v ph bng protein v ph bng ADN gia hai mu c B. similaris thu thp c hai vng M v Ct Tin. Tuy vy, s khc bit trong ph bng protein, ph bng ADN c lin quan vi nhau hay khng v c lin quan n s khc bit v hot ca 3 enzym SOD, NOX v CAT c iu tra hay khng l vn th v cn c tip tc nghin cu. TI LIU THAM KHO 1. Albayati, Z. A., Wahba, Z. Z., Stohs, S. J., 1988. 2,3,7,8-tetrachlorodibenzo -pdioxin (TCDD)-induced alterations in lipid peroxidation, enzymes and divalent cations in rat testis. Xenobiot. 18:1281-1289. 2. Albert, P. Senft., Timothy, P. Dalton., Daniel, W. Nebert., Mary, B. G., Richard, J. Hutchinson., Howard, G. Shertzer., 2002. Dioxin increases reactive oxygen production in mouse liver mitochrondria. Toxicol. Appl. Pharmacol., 178: 15-21. 3. Aruoma, O. I., Halliwell, B., 1991. DNA damage and free radicals. Chem. Br., 2:149-152. 4. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and assay applicable to acryamide gels. Anal. Biochem., 44: 276-287. 5. Farr, S.B. and Kogoma, T. 1991. Oxidative stress responses in Escherichia coli and Salmonel typhimurium. Microbiol. Rev. 55:561-585. 6. Hermanski, S. J., Holcslaw, T. L., Murray, W. J., Markin, R. S., Stohs, S. J., 1988. Biochemical and functional effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the heart of female rats. Toxicol. Appl. Pharmacol., 95: 175-184. 7. Leammli, V. K., 1970. Cleavage of structure protein during the assembly of the head of bacterophage T4. Nature., 227: 680-685. 8. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall. R. J., 1951. Protein measurement with the folin phenol reagent. J. Biochem., 193: 265-270. 9. Mayuree, F., Andrew, F. H., Nada, B., John, D. H., 2002. Regulation of the Bacillus subtilis of the PerR regulon are peroxide inducible. J. Bacteriol., 184(12): 3276-3286.

10. Mayuree, F., Andrew, F. H., Nada, B., John, D. H., 2002. Regulation of the Bacillus subtilis of the PerR regulon are peroxide inducible. J. Bacteriol., 184(12): 32763286.15. Mayuree, F., John, D.H., 2002. The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative. Proc.Natl.Acad.Sc 99 (10): 6690-6695. 11. Mc Cord, J. M., Fridovich, I., 1969. Superoxid dismutase an enzymic function for erythocuprein. J. Bio. Chem., 244: 6049-6055. 12. Mottola, H. A., Simpson, B. E., Gorin, G., 1970. Absorptiometric determination of hydrogen peroxide in submicrogram amount with leuco crystal violet and peroxidase as catalyst. Anal. Chemistry., 42: 410 411. 13. Park, J. Y., Shigenaga, M. K., Ames, B. N., 1996. Induction of cytochrome P450A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo (3,2-b) carbazole is associated with oxidative DNA damage. Proc. Nat. Acad. Sci. USA., 93: 2322-2327. 14. Phan Tun Ngha, Trn Cng Tc, Hunh Th Thu Hu v Phm Vn Lp, 1999. Tp ch Di truyn v ng dng. S 3: 56-60 15. Poole, L.B. and Claiborne, A. 1986. Interactions of pyrimidine nucleotide with redox forms of the flavin-containing NADH peroxidase form from Streptococus faecalis. J. Biol. Chem. 261:14525-14533. 16. Poland, A., Knutson, J. C., 1982. 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol., 22: 517-554. 17. Thibodeau, E. A., Keefe, T. E., 1990. pH-dependent fluoride inhibition of peroxidase activity. Oral Microbiol. Immunol., 5: 328-331. Summary COMPARISION OF ACTIVITIES OF SOME ENZYMES OF PROTECTION AGAINST OXIDATIVE DAMAGE AND DNA BANDING PATTERNS OF Bradybaena similaris COLLECTED FROM MA DA AND CAT TIEN (DONG NAI PROVINCE) The activities and thermostabilities of 3 enzymes: superoxide dismutase (SOD), NADH oxidase (NOX) and catalase (CAT) involved in protection against oxidative damage and the protein and DNA banding patterns of Bradybaena similaris collected from Ma Da and Cat Tien (Dong Nai Province) were compared. It was found from the study that the activities of SOD, NOX and CAT of Bradybaena similaris collected from Cat Tien were (1.8, 1.2 and 1.25 times respectively) higher than those collected from Ma Da. The protein profiles of the samples from both sites appaered to be similar, however, one band of 21 kDa was found only in the sample collected from Ma Da. The DNA banding patterns of the both samples were very similar; however, one band of approximately 500 bp was present only in the MA Da sample, whereas one band of approximately 750 bp was found only in the Cat Tien sample. The results suggested that futher investigation of the differences in activities of the studied enzymes and the protein and DNA banding patterns is needed to elucidate the effects of the environment on the system.

Ngi thm nh ni dung khoa hc: GS. L nh Lng.

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