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 FO L IA B 1 0 C H IM 1 C A E T H IO P H Y SIC A 9, 1992 Z o fia Szweda-Lewandowska E F F E C T O F W A T E R A N D E T H A N O L R A D IC A L S O N T H E P R O T E IN P A R T O F H U M A N H E M O G L O B IN P A R T II. D A M A G E T O A M IN O A C ID R ES ID U E S
T h is w ork is de vo te d to the effect o f in te rac tio n s o f w ater a n d e th a n o l rad icals w ith h u m an h em og lob in (H b ) a m ino a cid residues. T he O H ra dicals c aus ed the g rea tes t d es tru ction o f h u m a n h e m o g lo bin a m in o acid residues. A m o n g them th e m os t sensitive to irra d ia tio n were cysteine try p to p h a n a n d histidine residues.
I. IN T R O D U C T IO N
This w o rk deals w ith th e d am ag e to hem oglobin am in o acid residues induced by the actio n o f p rim ary p ro d uc ts o f w ater radiolysis and o f secondary ethano l radicals.
The effect o f the reactio n o f w ater and eth an o l radicals w ith the am in o acid residues was estim ated on the basis o f the loss in their co n ten t m easured on an au tom atic am ino acid analyzer afte r p ro tein hydrolysis. T he o btain ed results were discussed tog eth er w ith th e fluorescence changes in p a rt I [11],
2. M A T E R IA L S A N D M E T H O D S
H em oglobin was o b tain ed an d purified acco rdin g to pro ced u re described in the first p ar t o f o u r stu dy [II], G lobin was isolated from irrad iated hem oglobin by rem oval o f the hem e m oiety a t acid p H . T he m eth od described by Rossi F anelli [7] em ploys acid-aceton e a t low tem p eratu re to split the hem e g ro up from the globin, which precipitates an d is sub sequently redissolved in w ater.
3. IR R A D IA T IO N C O N D IT IO N S
Em ployed irrad ia tio n co nd itio n s were also ano log ou s as previously desc-ribed [11]. The p re p aratio n s were irra dia ted with 2.5 kG y and 5 kG y und er the atm osp h ere o f N 20 and arg on or in b oth varian ts with eth an ol. D eoxyhem og-lobin con cen tratio n was 5 g x dm 3.
4. A M I N O A C ID A N A L Y SIS
A m ino acid co m p osition o f hem o globin was determ ined follow ing protein hydrolysis in three variants:
in hy dro chloric acid at the co nc entratio n 5.7 mol x dm \
in p-toluenesulfo nic acid (2 x crystalized) at the co nc entration 3 mol x dm 3 with 0 .2 % 3(2 -am ino eth yl)ind ol from Pierce [5],
in m eth anesulfon ic acid at the con ce ntratio n 4 mol x dm 3 with 0.2% tryp tam ine from Sigm a [8].
T he am ino acid com positio n o f irrad ia ted H b an d globin o btained from irrad ia ted H b were analysed. The sam ples co ntainin g from 1 to 1.5 mg o f p rotein were hydrolysed in th e atm osp here o f argo n in sealed am pules at 110°C fo r 24 h. A m ino acid analysis w as perform ed using the au to m atic analyzer JL C -6AH (JEO L Japan).
5. T H E -SH G R O U P S D E T E R M IN A T IO N
T he -SH g ro up s co n ten t w as estim ated by sp ectro ph o to m e tric titration o f protein with p-chlorom ercuribenzoic acid (P C M B) [1], Th e PC M B p rep aratio n from C hem apo l was purified before use. T itratio n was perform ed using PCM B o f the co nc en tration 10 4 mol x dm 3 prepared in the acetate buffer, pH 4.6. Sam ples con tain ing 0.5 x 10 4 m ol x dm 3 o f pro tein were titrated . The p ro tein with PC M B was incub ated for 2.5 h in buffer pH 4.6 and then absorp tio n increase was m o nito red at 255 nm , an d the co nten t o f free -SH group s was calculated. The -SH gro u ps were determ in ed in the hem oglobin solu tion s exept the p re p aratio n s irra d iated with 5 kG y in the atm osph ere of N20. In this case the -SH gro up s were reco rded in solu tio ns o f globin ob tain ed from irrad iated hem oglobin.
6. R E SU L T S
The am in o acid residues co n ten t was m easured in irrad iated H b an d globin o b tained from irrad iated H b solutions. S im ilar results in b oth cases indicated the absence o f d etectable am o u n ts o f free am in o acids in the exam ined
A m in o a c id c o m p o s it io n in c o n tr o l a n d ir ra d ia te d h u m a n h e m o g lo b in w it h th e d o se 5 k G y A A -a m in o a c id C o n tr o l n 2 o N 20 + E t A rg o n A rg o n + E t m o l A A /m o l H b % A A m o l A A /m o l H b % A A m o l A A /m o l H b % A A m o l A A /m o l H b % A A m o l A A /m o l H b % A A L y si n e 4 4 “ 4 2 .6 ± 2 .9 b 1 0 0 3 9 .5 ± 0. 64 »> 9 2 .7 4 1 .6 ± 1 .6 b 9 7 .7 4 1 .9 + 1 .9 3 b 9 8 .4 4 2 .4 + 0 .8 4 b 9 9 .5 H is ti d in e 3 8 3 5 .8 ± 1 .3 9 1 0 0 2 9 .3 ± 0 .6 4 8 1 .8 3 9 .3 ± 5 .4 9 3 .0 ' 3 4 .4 ± 3 .8 7 9 6 .1 3 4 .8 ± 1 .2 9 9 7 .2 A rg in in e 1 2 1 1 .2 ± 0 .2 6 1 0 0 1 0 .5 7 + 0 .1 3 9 4 .3 1 1 .0 8 ± 0 .3 9 9 8 .9 1 1 .2 + 2 .5 8 1 0 0 .0 1 1 .3 + 0 .2 6 1 0 0 .8 A sp a rt ic a c id + a sp a rg in e 5 0 4 7 .9 ± 1 .9 9 1 0 0 4 6 .4 ± 1 .0 9 9 6 .9 4 9 .8 ± 3 .8 7 1 0 3 .9 4 8 .3 ± 1. 3 1 0 0 .8 4 9 .2 + 1 .9 1 0 2 .7 T h re o n in e 3 2 2 8 .4 ± 0 .1 3 1 0 0 2 4 .8 + 1 .1 6 8 7 .3 2 8 .1 ± 1 .9 9 8 .9 2 7 .5 + 1 .9 9 6 .8 2 8 .2 + 1 .9 9 9 .3 S e ri n e 3 2 2 8 .3 5 ± 2 .7 1 0 0 2 3 .6 ± 2 .5 8 8 3 .2 2 7 .4 ± 1 .5 5 9 6 .6 2 6 .2 + 3 .2 9 2 .4 2 5 .3 + 2 .6 8 9 .2 G lu ta m ic a c id + g lu ta m in e 3 2 3 1 .3 ± 1 .9 9 1 0 0 2 9 .1 ± 0 .5 8 9 2 .9 3 1 .6 ± 2 .5 5 1 0 0 .9 3 1 .0 ± 2 .6 9 9 .0 3 1 .9 ± 0 .7 7 1 0 1 .9 P ro li n e 2 8 2 6 .9 ± 1 .3 1 0 0 2 3 .1 + 0 .2 6 8 5 .6 2 6 .3 ± 1 .8 7 9 7 .8 2 7 .0 + 0 .4 5 1 0 0 .3 2 7 .0 + 1 .9 1 0 0 .3 G ly c in e 4 0 3 8 .7 ± 4 .5 1 0 0 3 5 .9 + 0 .9 0 9 2 .8 3 8 .7 + 0 .7 7 1 0 0 .0 3 8 .8 + 0 .9 0 1 0 0 .2 3 8 .2 + 1 .9 9 8 .7 A la n in e 7 2 6 9 .6 ± 1 .9 3 1 0 0 6 3 .7 ± 1 .9 3 9 1 .5 6 9 .6 ± 0 .8 4 1 0 0 .0 6 7 .7 ± 1 .9 9 7 .2 6 7 .7 + 2 .6 9 7 .2 V a li n e 6 2 5 9 .7 ± 2 .7 7 1 0 0 5 3 .9 ± 1 .9 3 9 0 .3 5 7 .6 ± 2 .6 9 6 .5 5 9 .0 + 2 .6 9 8 .8 5 7 .7 ± 0 .3 9 6 .6 M e th io n in e 6 5 .4 ± 0 .6 4 1 0 0 4 .7 7 ± 0 .6 4 8 8 .3 5 .0 5 ± 9 3 .1 5 .3 ± 1 .3 9 8 .1 5 .6 + 0 .4 5 1 0 3 .7 L e u c in e 7 2 6 9 .6 ± 3 .9 1 0 0 6 3 .7 + 1 .2 9 9 1 .5 7 0 .2 5 ± 3 .5 1 0 0 .9 6 8 .3 + 1 .3 9 8 .1 6 8 .3 + 2 .6 9 8 .1 T y ro si n e 12 1 0 .8 ± 0 .7 7 1 0 0 1 0 .1 + 0 .2 5 9 3 .5 1 0 .4 9 6 .3 1 0 .6 ± 0 .5 9 8 .1 1 1 .1 ± 0 .6 1 0 2 .8 P h e n y la la n in e 3 0 2 8 .9 ± 1 .5 4 1 0 0 2 6 .0 3 ± 0 .5 1 9 0 .1 2 8 .4 ± 3 .9 9 8 .3 2 7 .6 ± 3 .9 9 5 .5 2 8 .9 ± 0 .9 1 0 0 .0 T ry p to p h a n 6 6 .0 1 0 0 -6 .0 1 0 0 .0 -C y st e in e ' 6 5 .9 9 ± 0 .2 9 1 0 0 4 .5 2 7 5 .2 6 .3 ± 0 .2 9 1 0 4 .8 5 .0 5 ± 0 .2 5 8 4 .0 6 .0 1 1 0 0 .0 N u m b e r o f m e a s u re m e n ts n = 3 -1 0 a D a ta a c c o rd in g to G . F e r m i a n d M . F . P e r u t z , A tl a s o f M o le c u la r S tr u c tu r e s in B io lo g y 2 . H e m o g lo b in a n d M y o g lo b in , C la re n d o n P re ss , O x fo rd 1 9 8 1 , p p . 4 -5 . b A v e ra g e s ta n d a rd d e v ia ti o n c a lc u la te d a t m e a s u re m e n ts n u m b e r n o t sm a ll e r th a n 5 . S ta ti s ti c a ll y s ig n if ic a n t d if fe re n c e s in re la ti o n to c o n tr o l w e re e s ti m a te d b y t -S tu d e n t te st . T h e u n d e rl in e d v a lu e s sh o w s ta ti st ic a ll y s ig n if ic a n t d if fe re n c e s a t a = 0 .0 5 . T h e c o n te n t o f a m in o a c id r e si d u e s w a s d e te rm in e d f o ll o w in g p ro te in h y d ro ly si s in h y d ro c h lo ri c a c id a n d p -t o lu e n e su lf o n ic a c id ; tr y p to p h a n in m e th a n e su lf o n ic a c id , a n d c y st e in e b y B o y e r’ s m e th o d w it h o u t h y d ro ly si s.
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hem oglobin so lutions w hich m ight have occured as the possible p ro d u cts o f po lypep tid e b ond s d eg radatio n. The decrease o f am in o acid residues co nten t was in terpreted as the dam age. The analysis o f the am in o acid co m p o sitio n o f c o n tro l H b an d irrad iated w ith th e dose o f 2.5 an d 5 k G y w ere carried o ut. A t the dose o f 2.5 kG y the m easurem ents were p erfo rm ed following p rotein hydrolysis in p-toluenesulfonic and m eth anesu lfon ic acid. In the latter case the hydrolysis enables to d eterm ine the try p to p h an con ten t. Cysteine was es-tim ated by titratin g the -SH gro up s w ith PCM B.
The level o f -SH gro u ps decreased by 25% (Table 2) and histidine by 11% in H b irrad iated with 2.5 kG y u nd er the atm o sp h ere o f N 20 w itho ut ethano l. In the p rep aratio n s irrad iate d w ith th e sam e dose u nd er the atm osph ere o f arg o n w itho ut ethan ol, an d arg on and N 20 with eth ano l there were no differences in the level o f am ino acids betw een irrad ia ted and co n tro l Hb.
Table I presents the co n ten t o f am in o acid residues in H b irrad iated with 5 kG y in relatio n to co ntro l an d literatu re d ata. These results clearly indicate th a t losses o f H b am in o acid residues co nten t m ainly occur in the p re para tio ns irrad iated in the atm osph ere o f N 20 an d d o n o t exceed 25% . Cysteine (25% ) and histidine (18% ) display the largest decrease o f co nten t while proline, serine an d threo nine only ab o u t 15%. The loss o f lysine, arginine, glu tam ic acid + g lutam ine, alanine, valine, tyrosine an d p heny lalan in e is from 5% to 10%. Cysteine residues co nten t also decreased in H b irrad iated un der the atm o s-phere o f argo n (16% ). Th e co nten t o f the rem aining am in o acid residues does n o t show statistically significant differences in relatio n to the con trol. The presence o f ethano l in the p rep a ratio n s irrad ia ted in N 20 and argo n totally reduces the looses o f am in o acid residues co ntent.
T a b l e 2
T he -SH g ro u p s c o n te n t in c o n tro l a n d irrad ia te d in N2O a tm o sp h e re h u m a n h e m og lob in
D ose [kGy] 0.0 0.83 1.25 2.5 5.0
-SH [m ol/m ol Hb] 5.99 ± 0 .2 9 5.83 ± 0 .2 7 4.85 ± 0 .1 3 4 .4 9 ± 0 .3 4 4.52
The -SH grou p s co n te n t o f H b ir ra d iated in N 20 were determ ined in a wider dose range (Table 2). The results indicate th a t at the dose o f 1.25 kG y ab o u t 20% o f the -SH g ro u p s d o n ot react w ith P CM B . Th e m axim um decrease o f th e -SH g ro u p s is ab o u t 2 5% a t 2.5 kG y an d rem ains u nchang ed up to the dose o f 5 kG y. W ithin the exam ined dose range at the greatest a b o u t 1.5 m ol -S H /m ol H b is m odified as the result o f the in teractio n o f H b w ith the
7. D IS C U SS IO N
T he m o difications o f am in o acid residues leading to their loss d urin g the analysis by m eans o f the analyzer are relatively insignificant (irradiation con dition s given) an d initiated m ainly by the O H radicals (N 20 atm osph ere). A t the dose 2.5 kG y in N 20 only histidine co nten t decreases while try p to p h an rem ains the sam e in spite o f the d a ta from p a rt 1 th at try p to p h a n fluorescence in guanidine hy d rochlo ride (6 m ol x d m 3) decreased by ab o u t 23% [11]. T ho u gh insignificant the fluorescence decrease still suggests m od ification s within indol ring which m ay no t have any influence on try p to p h an beh aviour d uring am in o acid analysis. T he differences can derive from the use o f different m ethod s. In this case fluorescence m easurem en t is a m o re sensitive in dicato r o f try p to p h a n residues destru ction th a n the analysis em ploying the am ino acid analyzer.
The m aesurem ents o f the free -SH gro up s co n ten t in the irrad ia ted H b show their lim ited reactivity with w ater radiolysis pro ducts. The H b m olecule con tain s six cysteine residues: two in the chain at the position p F9 and
(> G 14 and one in the a chain a G 1 1 [6], The /i F9 cysteine residues are close to
the m olecule surface reacting w ith the reagents to th e -SH gro up s in native Hb. The a G 1 1 an d /? G 14 designated as „m ask ed ” react with the reagents to the -SH gro up s after protein d én atu ratio n .
It is w o rth notin g th a t app rox im ately 4.5 -SH g ro u p s/tetra m er H b were detected in the p rep a ra tio n s irrad ia ted w ith 5 kG y un der the atm osph ere o f N20 . A t this dose H b was an insoluble precy pitate and the -SH gro ups d eterm ina tion was carried o u t follow ing globin p recipitatio n. Such globin is well dissolw ed in acetate buffer, pH 4.6. The presence o f ab ou t 4.5 -SH gro ups per H b m olecule reacting with P CM B afte r globin unfo lding shows th at they d o n o t react w ith O H radicals un d er the irrad ia tio n con dition s assum ed by us. C ysteine is highly sensitive to w ater radicals, and the -SH g ro up s usually co n stitu te the active sites o f p roteins. Th e presence o f 4.5 -SH gro up s in the H b m olecule indicates th at u nd er irr ad ia tio n co n ditio ns used the H b m olecules are not dissociated in to su bun its to a g re at ex tent after th eir interaction with OH radicals and the protein core o f the m olecule practically stays beyond the direct radical action. T akin g into acco un t the results o f previous p ap er [9] one can suggest H b unfo ld ing in the m olecule surface regions con tain ing a helices with the tr y p to p h an residues A12.
A p a rt from try p to p h a n residues d estruc tion and the -SH grou ps destru c-tion at the dose 2.5 kG y in N 20 also histid in e co n ten t was foun d to decrease. H b co n tain s 38 residues am ong which 20 are exposed to the reaction s with hyd rogen ions in the pH range 6 -8 [2] and hence they are very likely to react with the 'O H radicals as well, yielding m o dificatio ns which affect histidine
co n te nt decrease. Losses in the co n ten t o f the rem aining am ino acid residues, n o t exceeding 15% , were stated at th e do se o f 5 kGy.
K u m ta and T appel [3, 4] analysed the am in o acid com positio ns o f H b solu tion irrad ia ted a t th e co n cen tration 1 g x dm 3 un d er th e atm osp here o f air and N 2. They applied p aper ch ro m a to g rap h y fo r am in o acid sep aration an d regarded the am in o acids co ntain ing su lp h u r next histidine, phenylalanine, serine and th reo n ine as m o st labile. T he decrease o f am ino acid residues co n ten t was obserw ed a t 10 kGy.
K eeping in m ind slight differences irrad ia tio n co n dition s in b o th studies it can still be concluded th a t am ino acid residues m o dificatio ns detectab le by the ap plied ch ro m a to g rap h y m etho d s occur at relatively high doses. The changes o f fluorescence an d ability to bind oxygen, o n the o th er h and , h ap pen at significantly lower doses [9, 11].
Besides o u r previous studies show ed th at O H radicals initiated agrég ation o f hem oglobin [10]. T he b o nd s induced by the actio n o f O H radicals are covalent. T ak in g into acco u n t changes in the am in o acid co m po sitio n induced by the O H radicals in dicate m od ification s o f cysteine, try p to p h a n and histidine residues. These m ay be targets o f O H radicals with H b and m ay be involved in the process o f aggreg ation.
A ckno w ledgem ent
T he a u th o r gratefully acknow ledges th e co n tin u o u s su p p o rt an d interest in this w ork by P ro fessor W. Leyko.
8. R E F E R E N C E S [1] B o y e r P. D. (1954), J. A m . C hem . Soc., 76, 4331-43 37. [2] J a n s e n L. H. M. , D e B r u i n S. H „ V a n o s G . A. J. (1970), B iochim . Biophys. A c ta , 221. 214 -227. [3] K u m t a U. S., T a p p e l A. L. (1961), „ N a t u re ” , 191, 1304 1305. [4] K u m t a D. S. , S h i m a z u F. , T a p p e l A. L. (1962). R a d ia t. R es.. 16, 679-685. [5] L i u T . Y „ C h a n g Y. H. (1971), J. Biol. C he m ., 246, 2842-2848. [6] P e r u t z M . F . (1965), J. M o l. Biol., 13, 6 4 6 668. [7] R o s s i F a n e l l i A. , A n t o n i n i E. , C a p u t o A. (1958), Biochim . B iophys. A c ta , 30. 608-615. [8] S i m p s o n R. J., N e u r e n b e r g M . N . R ., L i u T. Y. (1976), J. Biol. C h em ., 251, 1936—1940.
[9] S z w e d a - L e w a n d o w s k a Z . (1986), R a d ia t. E n viron . B iop hys., 25, 201 212. [10] S z w e d a - L e w a n d o w s k a Z. , P u c h a ł a M. , O s m u l s k i P. A. . R o s i n J. (1989),
R a d ia t. E n viron . B io phys., 28, 47 -58 .
[11] S z w e d a - L e w a n d o w s k a Z., P u c h a ł a M . (1989), S tud ia B iophys., 133, 123-131.
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Z o fia Szw eda -Lew and ow sk a
W P Ł Y W R O D N IK O W Y C H P R O D U K T Ó W R A D IO L IZ Y W O D Y I R O D N IK Ó W E T A N O L O W Y C H N A B IA Ł K O W Ą C Z Ę Ś Ć H E M O G L O B IN Y C Z Ł O W IE K A
C Z . II. U S Z K O D Z E N IA R E SZ T A M IN O K W A S O W Y C H
W pracy za jm o w an o się us zko dz eniam i reszt a m in ok w as ow y eh h em og lo b iny ind uk ow a ny m i d ziałan iem rod n ik ó w h yd ro ksylow ych , h y d ra to w a n yc h e le k tron ó w i ro dn ik ó w e tan o lo w yc h . J a k o k ry te riu m usz kod zenia prz yjęto obn iże nie (u by te k) w za w a rtoś ci reszt a m in ok w as ow y eh w n a -p ro m ie niow an y ch -p re -p a ra ta c h he m og lobiny. Z a w a rto ś ć reszt am ino kw a sow y eh o zn ac z an o -po hyd rolizie bia łk a za p o m o c ą a n a liza to ra am ino kw a sów . Poz iom g ru p -SH ok re ś la n o m e to d ą m iare czk o w a nia p -c hlo ro rtę ciob enz oe san em .
N ajba rd ziej efektyw n e w usz kod zeniu reszt am ino kw a sow yeh H b były ro d n ik i O H , n ato m ias t ja k o na jb a rdz ie j p rom ie nio czu le w b a da ny c h w a ru n k ac h n a p ro m ien ia n ia m oż na w ym ienić reszty cysteiny, try p to fa n u i histydyny.
