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 1M IC A E T B IO P H Y S IC A 9, 1992
Jolanta Gromadzinska, Bronislaw A. Zachara
A S E L E N O E N Z Y M E , G L U T A T H IO N E P E R O X ID A S E F R O M H U M A N P L A C E N T A : P U R IF IC A T IO N A N D S O M E P R O P E R T IE S *
G lu ta th io n e pe ro xid as e (G S H P x ) from h u m a n p la c e n ta w as pu rifie d a p p ro x im a te -ly 2200 tim es. A m m o nium su lfate (2 5 -6 0% s a tu ra tio n ) p re c ip ita tio n, S eph ade x gel filtra tio n an d D E A E -S e ph a de x A 50 c h ro m a to g ra p h y were used fo r pu rific ation p ro ce du re s . T he enzym e w as show n to be h om o g en o u s by po lyac ryla m id eg el e lec tro -phore sis. T h e m o le cu la r w eight (78 000 d a lto n s), isoelectric p o in t (5.50), o p tim u m pH (7 .8 -8 .0 ) an d tem p e ra tu re (42°C ), were d ete rm ine d. Io d o a c e ta te, N -e thy l m aleim id e and cy anide in hib it the enzym e activ ity by 80 90 % . D ith io th re ito l, G S H a n d 2-m ercap - to -eth a n o l p ro te c t the enzy m e from ina ctiva tion . D iva len t c a tio n s inh ib it th e a ctivity o f G S H -P x .
1. IN T R O D U C T IO N
G lu ta th io n e pero xid ase (G S H -Px; glutathion e: hy drog en perox ide oxidore- ductase; EC 1.11.1.9) discovered in 1957 by Mills [15] is up to now the only kn ow n m am m alian seleniu m -depend ent enzym e [5, 20], G S H -P x catalyses the redu ctio n o f H2O2 an d organic hy dro pero xides to w ater and correspo nd ing alcohols with the redu ctio n o f G S H . It is a p rim ary cellu lar an tiox idative enzym e and serves to prevent the form atio n o f toxic oxygen species. A ttem pts were m ad e to purify this enzym e from h um an [1], bovine [24] and o th e r anim al red cells [13, 19, 20] o r tissues [2, 16]. G S H -P x consists o f 4 identical sub units o f 21 000 d alton s and co ntain s 4 ato m s o f selenium per nativ e enzyme m olecule [5], Selenium is present at the active sites as selenocysteine residue [6] localized a t p ositio ns 4 1-4 7 from N -term inal end o f the protein [22], The refined th ree-dim ensio nal structu re o f the enzyme has been studied by Ep p et al. [4] an d the am in o acid sequence as well as the m echanism o f selenium in c orp o ra tio n has been elucidated [9, 17]. It has been show n [10] th a t hu m an
* Prese nte d a t th e 191*1 M e etin g o f the F ed e ra tio n o f E u ro p e a n B iochem ical Societies, R o m e (Italy), Ju ly 2 -7 , 1989 (A b s tra ct B o ok, p ap er N o. T H 176).
p lacen ta does n o t co nstitu te a b arrier to the m ovem ent o f seleno -am ino acids and accu m u lated seleno-m ethio nine, the n atu ra l organic form o f selenium . Ino rgan ic selenium and seleno-m ethionine are utilized in vario us m am m alian tissues fo r G S H -P x synthesis [23].
T he aim o f the present study w as to isolate the G S H -P x from h um an p lacenta an d to co m pare som e o f its p ro perties w ith those o f the enzyme isolated from o th er m am m alian sources.
2. M A T E R IA L S A N D M E T H O D S
2.1. Chemicals
S ephadex an d D EA E -S ephadex were o btain ed from P h arm acia Fine C hem icals AB, U pp sala, Sweden, G S H reductase, io do acetate an d E D T A were p u rch ased from Sigma Chem . C o., U SA. G S H , acrylam ide, t-butyl- an d cum ene h ydrop erox ide were kindly supplied by K och -L ig ht Labs, E ngland. All o th er chem ical used were o f analytical grade purity.
2.2. Purification procedures
All p urification procedu res were cond ucted at 0 4°C. F resh hu m an placen tae were used after a physiological delivery from health y wom en. The tissue was cu t in to pieces, washed in cold buffer con tain in g 0.9% N aC l an d 5 m M Tris-H C l, pH 7.6 (1: I; vol: vol) and hom ogenized. The h om o g en ate was centrifu ged a t 3000 g fo r 10 m in, the su p e rn a ta n t w as transferred and centrifu ged at 100 000 g for 1 h. T o th e clear su p e rn ata n t solid ( N H ^ S C ^ was ad ded. Th e p recipitate o b tain ed in the range o f 2 5 -6 0 % satu ratio n was used for fu rth er p urificatio n steps. P recipitated p rotein was dissolved in a small volum e o f 10 m M T ris-H C l buffer, p H 7.6 and dialysed again st the sam e buffer for 24 h. T he enzym e was centrifug ed an d the su p e rn atan t w as applied into a Sephadex G -100 colum n (2.5 x 100 cm) an d eluted with a Tris-H C l buffer, p H 7.6. A dditionally, the follow ing p urificatio n p ro cedu res were used [1, 25, 26] with slight m odifications:
a) D E A E-S ephadex A-50 colum n (2.5 x 30 cm ) and 0.05 -0.50 M N aC l in 10 m M T ris-H C l buffer, pH 6.5 as eluent.
b) G -150 colu m n (2.5 x 100 cm) an d 10 m M Tris-H C l buffer, pH 7.6 as eluent.
c) D E A E -S eph adex A-50 colum n (1 x 20 cm) and the sam e buffer as in a) d) ad d itio n al ch ro m a to g rap h y an d the sam e ion -ex changer an d buffer were used. P o ints a), c) and d) are num bered as D EA E -S ep had ex A -5 0 I, II and III.
2.3. Polyacrylamide gel electrophoresis
Disc gel electropho resis w as perform ed by the m etho d o f Laemli [12]. The isoelectric p oin t was determ ined according to the m eth od o f G ro n o w an d G riffith [8] using 4 % polyacrylam ide gel with 2.4% o f A m p holin e, pH 3-6. The protein was stained with C oom assie Brilliant Blue.
2.4. G SH -Px activity and protein concentration determinations
G S H -P x activity was assayed by the m eth od o f Paglia and V alentine [18] with t-butyl hy drop erox ide as sub strate. P ro tein co n cen tratio n in the eluates was determ ined sp ectrop ho tom etrically, an d ad dition ally in the G S H -P x peaks by the m eth od o f Lowry et al. [14],
3. R E SU L T S
3.1. Purification o f G S H -Px
T he results o f a typical p urification p ro ced ure are sum m arized in T able 1. The initial G S H -P x activity o f the am m o niu m sulfate fraction varied between 0.013-0.060 enzym e units. The results achieved in p ar tic u lar experim ents
T a b l e 1
Su m m ary o f p u rific atio n p ro c ed u re o f g lu ta th io n e peroxid ase
Step P ro te in co n cn (m g) T o ta l a ctiv i-ty* (u nits ) Specific activity P urificatio n (fold) Yield (% ) S u p e rn a ta n t 7340 235 0.032 1 .0 10 0 (N H4)2S 04 (2 5 -6 0 % ) 929 84.5 0.091 2 .8 69.3 Se ph ade x G -100 62 54.3 0.874 27.5 23.1 D E A E -Se p ha d ex A -50 I 15.1 46.5 3.08 97.1 19.8 Sep had ex G -150 1.6 40.3 25.2 793 17.2 D E AE -Sephadex A-50 II 0 .6 30.1 50.3 1587 1 2 .8 D E AE -Sephadex A-50 111 0.1 7.0 70.4 2 2 2 1 8.9 * O ne u nit o f G S H -P x is th e a m o u n t o f N A D P H ox id iz e d/m in /m g p ro te in (25°C).
varied very con sid erably. The degree o f final purification o f the enzym e varied from 1660 to 2550 times with the specific activity between 22.3-118.3 U /m g p ro tein an d the yield equalled 3 -17% . A typical ch ro m ato g rap h ic elution p attern o f G S H -P x from Sephadex G -150 and D E A E -S ep had ex A-50 III is presented in Fig. 1. As can be seen from this figure (up per p art), in a wide ab so rb an ce at 280 nm there is a sh a rp peak o f G SH -P x. The lower p ar t o f the figure show s th at the G S H -P x fraction is alm o st ho m ogenous. This fractio n o f G S H -P x was used in polyacry lam ide gel electroph oresis for checking th e h om ogeneity an d the m o lecular weight (Fig. 2). T he G S H -P x p rep a-ration m oved in po lyacrylam id e disc gel electroph oresis as a one protein band .
F r a c t i o n N u m b e r
Fig. 1. Se p ha d ex G 1S0 gel filtra tio n (u pp e r p a rt) a n d D E A E Se ph ad ex A 50 (III) c h ro m a to g ra -ph y (low er p a rt) o f h u m a n p lac en tal G S H -P x
Fig. 2. Po ly a cry lam ide disc gel elec trop ho re sis o f purified G S H -P x from h u m a n placen ta (left) an d s ta n d a rd p ro te in s (right). F ro m to p to b otto m : bo vin e liver c atalas e (240 000), ra b b it m uscle a ldo la se (160 000), ra b b it m uscle de h yd ro ge na se (144 000), h um an serum a lb u m in (67 000), o v o a lb um in (45 000) a nd c y to c hro m c (12 500). T h e m olec u la r w eight o f th e is olate d p lac en ta l
G SH -Px is 78 000
3.2. Properties of placental glutathione peroxidase
T he m olecu lar weight o f the purified enzym e, as d eterm in ed by poly ac-rylam ide gel disc electrophoresis, was 78 000 d alton s. Th e isolated enzyme focused as a single peak a t pH 5.50. The activity o f the G S H -P x was m easured at a pH rang e o f 6.2 -8.6 an d a t tem p eratu res between 25 50°C. T he o ptim um pH w as established to be 7.8 -8.0, an d th e o ptim u m te m p eratu re, 42°C. The enzym e app eared to be specific for reduced glutathio ne, as replacem ent by L-cysteine an d cysteam ine showed only 9.1 and 0.9% activity, respectively. A m o ng the hy d ro perox ides tested the activity observed was as follows: H2O2
> cum en e h yd rop ero xide > t-butyl hy drop ero xide (100. 85.2 and 69.6% , respectively). T he K m established for t-butyl hy drop ero xid e was 78.5 /tM . We observed th a t the purified G S H -P x was m uch less stable as co m p ared to the crude enzym e p re p ara tio n . The ad d ition o f d ith iothre ito l (D T T) o r G SH stabilizes the enzym e b oth at + 4 ° C and at -30°C [7], D ith io th reito l exerts
b etter effect an d th u s 0.2 m M D T T was ro utinely ad de d to the b uffer solu tio n used d u rin g the pu rificatio n procedures. On in cub ation o f purified G S H -P x w ith 10 m M io do acetate, N -ethyl m aleim ide an d cyanide th e enzym e is inactivated by 8 0 -9 0 % . W hen the dialysed enzym e was preincrtbated for 10 m in w ith D T T, G S H o r 2-m ercap toeth an ol and then in cub ated for 60 m in with 33 m M K C N , its activity did n ot ch ang e in the presence o f D TT , an d decreased by 2 1% in the presence o f the o th er tw o thiol stabilizing com p ou nd s.
In cu b atio n o f G S H -P x with 5 /¿mol o f divalent catio n s shows th a t H g + + and C u + + com pletely inactivate the enzyme, while Ni + + , C o + + an d C a + + inactivate it by 61, 53 an d 23% , respectively.
Because o f the enzym e instability and because o f the insufficient m aterial after the final pu rification step, the protein -to-selen ium ratio cou ld n o t be determ ined .
4. D IS C U SS IO N
G lu tath io n e p eroxidase purified from vario us sources w as show n to co ntain 4 mol o f selenium per mol o f enzym e [1, 2, 5, 24], Th e m olecular weights o f purified glutathio n e peroxidases varied betw een 76 000 97 000 d alto n s [1, 2, 11, 21, 24J. To the best o f o u r know ledge only one re p o rt has been pub lished on the G S H -P x m olecular weight, purified from bovine lens, to be as heigh as 140 000 d alto n s [3]. The p urificatio n m eth od s used in o u r experim ents yielded a 2200-fold pu rificatio n o f the enzym e. T he degree o f p urificatio n described by o ther a u th o rs w as in m ost cases alm o st in th e sam e ran ge [13, 24, 25], D ifferent m olecular weights, and v ariou s p u rifica tion folds o f th e ho m o gen ou s fractio n were p rob ab ly due to d ifferen t analytical techn iques used in th eir experim ents. Th e enzym e purified by us had a specific activity o f 70.4 U /m g p ro tein, which is higher th an th a t purified from bovine lens [11] b u t significantly lower th an the enzym e purified from hu m an ery th rocy tes w ith specific activity o f 103 U /m g p ro tein [1] o r th a t purified from o th er sources w ith m uch higher specific activities [2, 3, 13, 21, 24-26]. The rep o rted stability o f the purified G S H -P x was highly variable. O u r resu lt is in accord w ith Little et al. [13] w ho observed th a t the less purified sam ples o f the enzym e were m u ch m ore stable to sto rag e th a n purified G S H -P x. A w asthi et al. [1] have show n th a t the ery thro cy te enzyme is stable a t p H 7.0 at 4°C bu t very u nstab le a t low p H values. A t p H 4.0 the enzym e inversely lost all the activity in 20 m in. Bergad et al. [3] dem o n strated th a t co n c en trated (m ore th a n
100 un its/m l) lens enzym e p rep a ra tio n s lost no activity for 6 m o n th s when sto red at - 2 0 to -8 0°C . How ever, diluted sam ples (less th a n 1 un its/m l) lost ab o u t 65% o f th eir activity when kept 3 h o u rs at -2 0°C . In the presence o f bovine serum albu m in the enzym e activity was stabilized.
R epo rts from several labo rato ries differ in the values o f isoelectric p H o f the purified G S H -P x. O u r finding d em o n strates th at the p i is 5.50, while A w asthi et al. [1, 2] have shown p i values o f 4.8 4.9 and Y osh im u ra et al. [25] fo und th at the pH o f rat liver enzym e is 6.96. Also th e repo rted pH op tim um for G S H -P x activity is highly variable: 7.9 and app rox im ately 9.0 for bovine lens [3, 11], 8.5 for erythrocytes, and liver [1, 24, 25], and 7.8 8.0 fo und in this study. These differences m ay be due to the technique used o r to the source o f the enzym e. The results presented here show th a t g lu tath io n e ij the m o st active su b strate for G S H -P x. O u r results show th at G S H -P x exhibits greater affinity for a n atu ral su b strate, H2O2 th an fo r o th er sub strates at co m p arab le co n cen tration s. These d a ta are in acco rd with o th er au th o rs [2, 3, 11, 26], A nu m b er o f lab o rato ries have re po rted inh ibition o f the enzym e following the ad d ition o f io d oacetate [1, 13, 18, 26], N -ethyl m aleim ide [1, 13, 18], K C N [2, 19] an d several divalent catio n s [1], The results o btained in o u r la b o ra to ry fully confirm the ab ov e m entioned ob servations.
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P E R O K S Y D A Z A G L U T A T IO N O W A S E L E N O Z A L E Ż N Y E N Z Y M Z Ł O Ż Y S K A L U D Z K IE G O : O C Z Y S Z C Z A N IE I N IE K T Ó R E W Ł A ŚC IW O ŚC I
P erok sy da zę g lu ta tio n o w ą (G S H -Px ) z łożyska ludzkie go oc zyszc zono ok . 220 0-k rotnie , stos ując n a stę pu ją ce techn iki: w ytrą ca nie siarcz ane m a m o no w ym (2560% na syc enia), c h ro m a to -grafia n a Se ph ade x G -100, S eph ad ex G -15 0 o ra z D E A E -S e ph ad ex A -50. E le k tro fo re za w żelu p oliak rylam ido w y m w y ka zała istnien ie je d n o ro d n e j frakc ji G S H -Px . W oczyszc zo nym enzym ie o z n ac z o no m as ę cz ąste cz kow ą (78 000 d a lton ó w ), p u n k t izo elektryczn y (5,50), o p tim u m p H (7 ,8 -8 ,0 ) i o p tim u m te m p e ra tu ry (42°C ). J o d o o c ta n , N -etyle no m a leim id i cy ja nk i inh ibu ją a ktyw n o ść enzy m u w 8 0 -9 0 % . D itio tre ito l, g lu ta tio n i 2 -m erk a p to e tan o l c h ro n ią enzym przed in ak ty w a cją. D w u w a rto ściow e k a tio n y są rów nie ż inh ib ito ra m i G SH -P x.
