A C T A U N I V E R S I T A T I S L O D Z I E N S I S
F O L IA B IO C H IM IC A E T B IO P H Y S IC A 9, 1992
W irgiliusz Duda, Paweł A. O sm ulski
S T U D IE S O N P A R A Q U A T A N D G R A M O X O N E IN T E R A C T IO N S W IT H H E M O G L O B IN S *
In this w ork th e o x id a tio n o f h u m an a n d c a rp he m o glob in s (H b ) by P a r a q u a t (PQ ) a n d G ra m o x o n e (G X ) w as stu died . W e d e m o ns tra te d th a t th e proce ss ru n s th ro u g h valency hy b rid s, m ain ly a 3 + /?2 + , to m et- a n d fe rry l-H b. T he re ac tion is m ed ia te d by „a ctive form s o f ox yg en": H jO i, O H a n d O^ It is c ha ra c te ris tic, th a t hy dro gen pe ro xide oxidises directly hem e iron to its ferryl fo rm w ith Fe4 + . T h e active fo rm s o f oxygen in th e c o o p e ra tio n w ith PQ ca use g lob in d é n a tu ra tio n an d d a m a ge o f H b - SH g ro u ps a n d T rp residues.
1. IN T R O D U C T IO N
P a ra q u a t (PQ ) as well as its 25% aq u eo u s solu tio n G ra m o x o n e (G X ) is system ic and co n tact herbicide with chem ical nam e 1, r-d im eth y l-4 , 4’-bipyri- d inium dichloride. It is widely used co m p o u nd being a t the sam e tim e highly toxic n o t only to all green plants bu t also to nearly all form s o f life from m icroorganism s to m am m als. Thu s, the u nd erstan din g o f m echanism o f its toxic actio n seem s to be a h o t topic. O n the cellular level an d from m edical p oin t o f view o u r knowledge o f its actio n is nearly full. O n the o th er h an d , on the m olecu lar level o u r know ledge is relatively po o r. Som e d a ta sho w th a t oxygen an d its free radical active fo rm s play the m o st im p o rtan t role in the developm ent o f p oiso no u s effects o f PQ and G X . T he c ur ren t stud y was u n d ertak en to clarify som e o f the m olecular m echanism o f PQ an d G X interaction with hem o glob in and to com pare the actio n o f these tw o com po und s.
* In th is p a p er re sults p res en ted in X ll-th In te rn atio n a l Sym po sium on S tru c tu re an d F u n c tio n o f E ry th ro id C ells, A u g us t 2 8-3 1, 1989. Berlin have been su m m arized .
2. M A T E R IA L S
1. H u m an H b A w as isolated using the m etho d o f Rigss, B o nav entu ra and B on aven tura [6] and purified acco rd ing to T en tori et al. [10].
2. C arp H b (H b C c - C yprin us carp io ) was isolated using the m eth o d o f Lin et al. [4].
3. Th e p ro tein s studied were oxidized w ith a b o u t tw o fold excess o f K3[Fe(CN)fi] which was subsequently rem oved by a S ephadex G-25 colu m n ch ro m ato grap h y .
4. PQ p.a. was pu rchased from Sigm a (U .S .A .), G X was o b tain ed as a com m ercial 25% aq ue ou s stock so lutio n o f herbicide fro m A grotek (H u ngary ).
5. All o th er chem icals were o f analytical grade an d used w itho u t fu rthe r purificatio.
6. D ouble-distilled, deionized w ater was used th ro u g h o u t the experim ents.
3. M E T H O D S
R eactio n co nditions:
- co nc entratio n o f H b 100 /im ol 1 1 (in respect to hem e - 1.6 m g/m l o f H b), - PQ o r G X co n cen tratio n 0 -50 /ig/m l o f H b,
- incu batio n d u ring 0 2 hrs in 0.2 m ol • 1 1 K -ph o sp h ate buffer, pH 6--8, at tem p eratu re 37°C.
A nalytical m ethods: - sp ectro ph o tom e try,
- cycle m easurem ents o f a b so rp tio n spectra in a UV-Vis range (S P E C O R D M 4 0 C arl Zeiss Jena).
---m u ltico m p o nen t analysis on the basis o f ab so rp tio n changes at ch a-racteristic wavelengths for different H b form s, using „C o m p u te r K assette D A T A H A N D L IN G II zum S P E C O R D M 4 0 ” ,
- ch ro m a to g rap h y after H b in cub atio n with the agents, H b form s were an alysed using colum n ch ro m a to g rap h y with CM -C ellu lose [10],
- d eterm in atio n o f free -SH grou ps [1], - try p to p h a n analysis [3].
4. R E S U L T S
O xy H b a b so r p tio n spectra after the PQ o r G X actio n showed th e decrease o f a an d /i b and s (540 an d 576 nm , respectively) and the increase at 500 and 630 nm (see Fig. 1, 2).
F ig . 1. A b s o rp ti o n sp e c tr a (c y c le ) o f H b C c in th e p re se n c e o f G X (1 0 p p m ) F ig . 2 . A b s o rp ti o n s p e c tr a (c y c le ) o f H b A in th e p re se n c e o f G X (1 0 p p m ) o
R eaction rates o f ox yH b decay dep en ded stro ngly on the pH con ditio ns. H ig her o xidatio n rates were fou nd a t low er pH in all th e system s investigated. H b C c was m ore susceptible p rotein on G X action th an the H b A . T heir oxidised derivatives created easier d en atu red form s th an their oxygenated derivatives (see T ab le 1, 2).
T a b l e 1
O bs erve d re ac tio n ra te s o f o x y H b de c ay a fte r PQ o r G X a c tio n (50 pp m )
Sys tem in ves tigate d PH R e ac tio n rate s (m in 1)
H b C c + PQ 7 (5.74 ± 0 .2 9 ) 10 4 + G X 6 (7.67 + 0.53) • 10 3 7 (3.91 ± 0 .3 4 ) ■ l( H 8 (7 .5 5 ± 0 .3 2 ) ■ 10 4 H b A + PQ 7 (2 .7 9 ± 0 .1 1 ) • 10 4 + G X 6 (8 .3 2 ± 0 .2 2 ) • 10 4 7 (5.63 ± 0 .2 0 ) 10 4 8 (2.83 ± 0 .1 5 ) • 10 4 d2A /dx2 A [n m ]
Fig. 3. T h e sec ond d eriv ative s o f a b s o rp tio n s pe c tra o f H b A in cu b ated w ith G X (50 p p m ) o r with H 20 2 (5 n g /m l H b )
t- [ min ]
Fig. 4. R ela tive c o n te n ts o f H b fo rm s d u rin g PQ a c tion (50 p pm ) o n H b C c, p H 7.0 (resu lts o f m u ltico m p o ne n t an alysis)
t [min ]
Fig. 5. R elative c o nte n ts o f H b fo rm s d u rin g G X a ctio n (50 p pm ) on H b Cc, p H 7.0 (resu lts o f m u ltico m p o n e n t a nalysis)
T a b l e 2
R elative c o n te n ts o f F e(III) (C % ), n o n -d e n a tu re d (N % ) a n d ox yge na ted (0% ) H b fo rm s a fte r 3 hrs. in cu b a tio n o f o x y H b with PQ o r G X (50 ppm ) System in vestiga ted pH C % N % 0 % H b C c + PQ 7 89 ± 5 51 ± 1 1 21 ± 7 + G X 6 9 9 + 5 32 ± 1 3 4 ± 10 7 98 ± 4 47 ± 9 9 ± 1 0 8 92 ± 5 69 ± 3 15 ± 7 H b A + PQ 7 56 ± 8 79 ± 6 4 7 ± 13 + G X 6 82 ± 9 8 4 + 1 3 21 ± 7 7 70 ± 5 88 ± 5 34 ± 8 8 41 ± 6 88 ± 3 5 4 ± 10 11 21 31 41 51 61 71 81
fra c tio n number
-oxyHb + Gx oxyHb/m etHb metHb + Gx
1 - oxyHb, 2 - o t2+ (33+ 3 - o<3+ (S2+, 4 - metHb
Fig. 6. E lu tio n profiles o f H bA in c u b a te d w ith G X (50 pp m ). In c u b a tio n tim e 2 hrs
PQ was m uch less effective in co m parison w ith G X . The presence o f 591 nm b an d in the second derivatives o f ab so rp tion spectra o f H b after its in cub ation with PQ, G X o r H2O2 suggests th a t there are also a n o th e r H b form s in the m ix tu re (see Fig. 3).
The ad d ition al form s which m ight be taken into acco un t were ferryl (F e4 + ) and hem ichrom es. M u ltico m p on en t analysis o f th eir kinetic changes was perform ed on the basis o f ab so r p tio n intensity at their characteristic peaks for all distinguished form s (see Fig. 4, 5).
This allow ed to m o n ito r th a t du rin g the oxy H b decays, m etH b level first increased, after then show ed a relative sh ort decrease an d subsequ ently increase to a n ear co n stan t value, lower th an 100% . The level o f ferryl form grew very slow an d after the m axim um transitio n it decayed quickly (see Fig.
6, 7).
t [min ]
Fig. 7. C ha ng es o f re lativ e c o n te n t o f H b A form s a fte r in cu b a tio n w ith G X (50 p p m ) - resu lts o f c h ro m a to g ra p h ic se p a ra tio ns
Ion exchange c h ro m a to g rap h y o f ox yH b A in cu bated w ith PQ o r G X exhibited the presence o f five m ain fractio ns in which free hem e, hem e-globin com plexes, o xyH b , a 3+ /j2+ valency hybrids, an d m etH b were eluted. A sim ilar p atter n o f fraction s was o b tained after the separation o f incubated m etH bA (with the exception o f ox yH b fraction ) - see T able 3.
E lu tio n a n d s p e c tro p h o to m etric p rop e rtie s o f fra ctio ns o b ta in e d a fte r c h ro m a to g ra p h ic se p a ra tio n o f o x y H b in c u b a te d 0.5 hr. w ith G X , a n d the e q u im o lar m ixture o f oxy- an d m e tH b A
F ra c tio n E lu tion PH <xlp ra tio So ret b an d (nm ) UV m ax. (nm ) S o re t/U V m ax. ra tio o xy H b A + 20 ¿ig G X /m l -lb, 0.5 hr., 370C • glob in 6.70 - - 268 _
h em e-g lobin com plexes 6.78 i . 85 1.000 414 272 2.88
o x yH b 7 .02 -7.0 4 1.042 412 275 3.57
a 3+ hy brids 7 .16 -7.2 0 0.933 407 275 3.24
m etH b 7.2 8-7.3 2 - 406 275 4.86
e q im o lar m ix tu re o f oxy- an d m etH b
o xy H b 7.02 -7 .03 1.040 412 275 3.58
a 2+ /?3+ hy brid s 7 .08 -7.10 0.964 410 275 3.93
a 3+ p2+ hy brid s 7.17-7.21 0.927 408 274 3.93
m e tH b 7.27-7.32 - 405 275 5.06
[ n m]
-S H / H b i e tr a m e r □ H b A + G x E E S H b A + P Q V M H bA c o n tr o l H bC c + G x E K ä H b C c + P Q ■ H b C c c o n tr o l p H F ig . 9 . N u m b e r o f -S H g ro u p s p e r H b te tr a m e r a ft e r 3 h rs a c ti o n o f P Q o r G X (5 0 p p m ) o n H b C c o r H b A 1 2 10 T rp / H b te tr a m e r %
i
ä
M w-v; ; I' i □ H b A < G x K T -B H b A •» P Q H b A co n tr o l S 3 H b 'C c + G x s s a H b C c + P Q ■ H b C c c o n tr o l B 7 7 p H F ig . 1 0 . N u m b e r o f T rp g ro u p s p e r H b t e tr a m e r a ft e r 3 h rs a c ti o n o f P Q o r G X (5 0 p p m ) o n H b C c o r H b AT he p rop er identification o f eluted fractions was d on e on the basis o f co m p arison o f elution an d sp ectro p h oto m etric p ro perties o f the o b tained fraction s w ith those resulting from eq uim olar m ixture o f oxy- an d m etH b sep aratio n. It has been show n th at the in teractio n o f the agen ts with H b led to p rotein d é n atu ra tio n (see Fig, 8).
The increase o f ab so rp tio n in ten sity at 275 276 nm (-x-, -o-) a n d the small shift (-0-) strongly su p po rt this idea.
The change o f spatial stru ctu re o f globin is also connected with destru ctio n o f the -SH and T rp g ro up s (see Fig. 9, 10).
A gain, G X was the m ost effective in this action, especially at pH 6. The -SH g ro u p s o f H b Cc seem to be the m ost susceptible. The T rp gro up s are destroy ed to sm aller extent. The highest stability o f globin an d ferrous heme iron was found at pH 8 in all the cases investigated.
5. D ISC U S S IO N
Inte ractio n o f P Q or G X w ith H b s leads to o xid ation o f their hem e iron to ferric an d ferryl form s. A t the first stage o f reaction a radical form o f bipyridyl is gen erated w hich after the reaction w ith m olecular oxygen creates superoxide radicals (1). They dism ute to hy drogen perox ide which seems to be a direct reason for the late ferryl p ro d u ctio n (2 4) [9, 2]. Results o f m ultico m p on en t analysis indicate th at fu rth er stages o f reaction s lead to the d en a tu red , oxidized derivatives o f H b, also th ro u g h d isp ro p o rtio n atio n o f ferryl and oxygenated form s (4, 5). T he direct actio n o f bipy ridyl m ay be connected w ith in tercalar bin ding to the globin an d (or) electro n tra nsfer between the ag ent an d po rp hy rin. P Q + + 02 - P Q 2+ + 0 ¿ (1) 2 0 i + 2 H + ^ H202 + 02 (2) H b 2+ + H20 2 H b 3+ + O H + O H (3) H b 3+ + H202 H b3 + H20 2 H b 4+ + O H + O H (4) H b 4+ + H b 2+ -*• 2 H b 3+ hem ichrom es (5)
The presence o f bipyridyl d eg rad a tio n p ro d uc ts m ay explain th e observed higher efficiency o f G X in the co m parison w ith the p ure PQ so lu tio n [5], H ow ever, a certain role in the d ev elop m ent o f toxic actio n m ay be played by
ions o f tran sition m etals (iron ) w hich also origine from Hb. They can w ork in F en to n o r F enton -like reaction s [7). N o t only the hem e iron is oxidized du ring the interactio n bu t also the p orp h yrin is destroy ed, the globin d en atu red and som e am in o acids degrad ed (especially Cys and less Trp). The process is alw ays b etter visible in the acidic pH . This follows generally the co nc ep t o f lower stability o f globin an d reduced heme iro n a t higher p ro to n co ncen tratio n s connected with ex position o f hy d ro p h ob ic protein co re and proto n-catalysed H b o xidatio n [8], The o b tain ed ch ro m atog rap h ic d a ta confirm the results o f sp ectrop h o tom etric investigations. They show in ad d ition th at the o xid ation goes th ro u gh the valency hybrids creation b u t practically only the a 3+ /i2 + ones were found. It seems th at these results strongly su p p o rt the free radical m echan ism w ith sequential o xidatio n o f the hem e iron thro u gh the ferryl fo rm atio n. The process is m ore p ro n o un ced in the presence o f m etal ions and the d eg rad ation p ro du cts o f PQ. T he p ro posed m echanism is p rob ab ly co m m o n for the in teractio n o f PQ w ith all p roteins contain in g m etallo por- phy rins [9],
6. C O N C L U S IO N S
*
1. PQ and G X induce o x idation o f H b to m etH b an d ferrylH b. T h e process ru ns th rou g h the valency hybrids, m ainly a 3+ [i2+ .
2. The reactio n is m ediated by the „activ e fo rm s o f oxygen” : H2O2, O H and O f . H y dro gen peroxide p ro ba bly oxidises directly hem e iron to its ferryl form.
3. The active form s o f oxygen in the coo p eratio n with PQ cause globin d é n atu ra tio n an d dam age o f its -SH an d T rp groups.
4. T he second deriv ative o f ab so rp tio n spectrum o f ferryl form exhibits characteristic m axim a which allow to distinguish the ferryl from o th er H b derivatives.
This w ork was su pp o rted by Research G ra n t Subject II. 11.1.1.
7. R E F E R E N C E S
[1] A l e x a n d e r N . M. (1976), [in:] R z u c i d ł o R ., Im m unologia ogólna i doświadczalna, P W N . W arsz aw a , 176-178. [2] B e l o q u i O. , C e d e r b a u m A. I. (1985), A rch . B iochem . B iophys., 242, 187-196. [3] C h r a s t i l J. (1986), A n aly s. B iochem ., 158, 443 446. [4] L i n M . J.-J ., N o b l e R. W. , W i n t e r h a l t e r K. H „ D i l o r i o E. E. (1988), Biochem . B iophys. A c ta , 954, 7 3-81 . [5] N a n n i E. J., A n g e l i s C. T. , D i c k s o n J., S a w y e r D. T. (1981), J Am . C h em . Soc., 103, 4268- 4270.
[6] R i g g s A. , B o n a v e n t u r a J. , B o n a V e n t u r a C . (1981), [in:] A n t o n i n i E. , R o s - s i B e r n a r d i L., C h i a n c o n e E. (E ds.), M eth ods in E nz ym olo gy, vol. 76, „ H e m o -g lo b ins ” , A c adem ic Press, New. Y o rk, 5 29.
[7] S a d r z a d e h S. M. , G r a f E., P a n t e r S. S., H a l l a w a y P. E., E a t o n J. W. (1984), J. Biol. C he m ., 259, 14 3 54-1 4 356.
[8] S a t o h Y. , S h i k a m a K. (1981), J. Biol. C he m ., 256, 10 175 10 272. [9] S m i t h L. L. (1985), Phil. T ran s . Res. Soc. L on d., B 3I1, 647-657.
[10] T e n t o r i L., V i v a I d i G ., C a r t a S., S a 1 v a t i A. M ., S o r c i n i M ., V a 1 a m i S. (1965), A rch . B iochem . B iophys., 100, 404 414.
C am e in e d itoria l office „ F o lia b io chim ic a e t b iop h y sica ” 26.06.1991
W irgiliusz Duda, P aw eł A. O s m uls ki
B A D A N IA IN T E R A K C J I P A R A K W A T U I G R A M O X O N U Z H E M O G L O B IN Ą
W p ra cy p rz e d sta w io n o w yniki b a d a ń n ad u tlen ian iem przez p a ra k w a t i g ra m o x on h em o glob in y czło w ieka i k a rp ia . J a k w y k az an o , p roc es ten p rzebiega z w ytw orzeniem h yb ry d w alenc yjnych H b głów nie a 3+ fi2+ i fe rrylh em o glob in y d o k ońc ow e go p ro d u k tu m etH b. T a re ak cja je s t w yw oływ ana p rze z a kty w ne fo rm y tlenu: O H , O2 i H2O2. W y ka z an o , iż c h a ra k te ry s -ty czne jes t, że H2O2 b ezp o ś red n io u tle nia żelazo he m u do form y ferrylow ej (tj. F e 4 + ). A kty w ne fo rm y tlen u (ro d nik i) p od w pływ em p a ra k w a tu lub g ram o x o nu p o w o d u ją rów n ież, w dłu ższym czasie d z ia ła n ia , czę ściow ą d e n a tu ra c ję glob in y prz y czym w ystępu je u szk odz enie g ru p -SH i reszt try p to fa n u.
C h a ir o f B iophysics U n ivers ity o f Ł ó d ź
