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 ____ FOLI A C H I M I C A 8, 1908
B o g u s ł a w Gr ze lak*, W i e s ł a w A u g u s t y ni ak **, Jan M. R o g o ziń sk i |***, S t a n i s ł a w R o m a n o w s k i *
SOME A T TE MPT S OF E L E C T R O C H E M I C A L O X I D A T I O N OF HU MAN BL OOD HE M O G L O B I N E ON THE GO LD E L E C T R O D E * * * *
C y cl ic c h r o n o v o l t a m p e r o m e t r i c m e t h o d was a p p l i e d in a t t e mpt s of o x i d a t i o n of d e o x y h e m o g l o b i n hu man b l oo d s o l u t i o n s in d i f f e rent a n t i c o a g u l a n t s on the gold el ec tro de . On the basis of ob t a i n e d results it was as su m e d that the o x i d a t i o n of h e m o g l o b i n in 3.8% sodi um ci tr a t e s o lu tio n and also in 1% NaCl and 0.5% NaF m i x t u r e s is possible.
H e m o g l o b i n e (Hb) - the main c o l o u r e d c o n s t i t u e n t of e r y t h r o c y t e s pl ays e x t r e m e l y im po rta nt role in h u ma n o r g a n i s m as .the o x yg en and carb on di ox i d e carrier. It is built up of four he m m o l e c u l e s with two pairs of p o l y p e p t i d chains:
H O O C C O O H
Mo l e c u l e of hem
*
De p a r t m e n t of Ge ne ral and In or gan ic Ch em ist ry , In st itu te of Ch em ist ry , U n i v e r s i t y of L6d2.
**
St at i o n of Bl ood Donors, MSW Hospital, D e p a r t m e n t of In ternal M e d i c i n e M i li tar y Medi cal Academy, L<5d2.
Clin ic of O i a b eto lo gy , Ac ad emy of Me di cin e, L 6 dt . * * * *
This p a pe r has been done and s u p p o r t e d in the f r a m ewo rk of the I n t e r d e p a r t m e n t Pr oj e c t MR I— 11.
B i v a l e n t iron in the p o r p h y r i n c o m p l e x is ab le to fix the o x y g e n w i t h o u t c h a n g i n g the v a l e n c e in m o l a r r a ti o 1 : 1 . O x y g e n a d d i t i o n to Hb c a ll ed o x i d a t i o n is not a c o m p l e t e l y r e v e r s i b l e process. In the o x i d a t e d co mp l e x very slow c h e m i c a l o x i d a t i o n of the b i v a l e n t iron takes p l ac e the e f fe ct of w h ic h is the c r e a t i o n of m e t h e m o g l o b i n e (MetHb) c o n s i s t e d of p o r p h y r i n c o m p l e x e s Fe (I II) c a ll ed hemines, he nce the anot her name of m e t h e m o g l o b i n e - he mi- gl ob i n e [ l , 33].
En zy m e s redu cin g M e tH b to Hb i n c l u d e d in bloo d c e ll s o p po se this p r o c e s s but the c o n t e n t of M e t H b in the o x i d a t e d bl ood a p p r o x i m a t e s 2%. G r e a t e r am mo unt of M e t H b c a us es the s t at e of s i c k n e s s m a i n l y b e c a u s e of the r e d u c e d v e n t i l a t i o n p r o p e r t y of h u ma n bloo d [1-5, 30, 31].
The s p e c i f i c c h a r a c t e r of Hb a b i l i t y of fixi ng the o x y g e n and also its very slow c h a n g e into M e t H b can be o b s e r v e d in the e x p e ri me n t s wi t h the o x y g e n o x i d a t i o n in the s y n t h e t i c h e m o - s i m i l a r co mp oun ds . P o r p h y r i n c o m p l e x e s Fe(I I) wi th p r o t e i n c h a i n s are o x yg en o x i d a t e d 1 0 8 times faster th an n a t u r a l Hb [6]. P i r i d i n i a n s o l u t i o n s of s y n t h e t i c p o r p h y r i n c o m p l e x e s with Fe(I I) n e i t h e r pr es e n t p r o p e r t i e s of c o n n e c t i n g w i t h o x y g e n nor they o x i d a t e to c o m p l e x e s w i th Fe(III). Only the a d d i t i o n of w a te r to the s o l u t i o n c a us es qu ick a u t o - o x i d a t i o n [5] .
The d e o x i d a t e d Hb ( d e o x i h e m o g l o b i n e ) very q u i c k l y a t t a c h e s the o x yg en fr om the air. 2, 3 - g l y c e r i n e d i p h o s p h a t e (2, 3-OPG) has a great i n f l uen ce on the Hb a f fi nit y for the oxygen. The d e c r e a s e of 2, 3-DPG c o n t ent in huma n b l oo d o b s e r v e d d u r i n g long b l o o d pre s e r v a t i o n in f l u e n c e s the i n c r eas e of Hb a f f i n i t y for the o x yg en and its mo r e d i f f i c u l t r e l e a s e ( r e d uce d v e n t i l a t i o n ) [30, 31],
The p r e s e n c e of o x i d a t e d or d e o x i d a t e d Hb can be e a si ly stat ed by c o m p a r i n g a d s o r p t i o n s p e c tra of Hb so lu tio ns . The o x i d a t e d Hb g i ves two clea r a b s o r p t i o n band s at 578 nm and 540 nm in the visible part of a b s o r p t i o n sp ec trum, w h i l e the d e o x i d a t e d Hb g i ve s only one at 555 nm [ l , 4] .
The o x i d a i i o n of Hb to M e tH b can be e a si ly c a r r i e d out th ro u g h the p o t e n t i o m e t r i c f e r r i c y a n i d e ti tr ati on . The n o rm al H b / M e t H b p o t e n t i a l in these c o n d i t i o n s d e p e n d s on pH and it c h a n g e s from 0.175 V (SHE) at pH * 6 to 0. 040 V (SHE) at pH = 9; pH of h u m a n blood is about 7.4. This r e a c t i o n is not 4 - e l e c t r o n as it s h o u l d have b e en s u s p e c t e d but n value i n c l ude s itself w i t h i n the limi ts from
1.1 to 2.7 in d e p e n d e n c e of pH [ l - 4 ] . It is p r o b a b l y c o n n e c t e d with the c h an ge b o th in the hem s t r u c t u r e and in the w h o l e h e m o g l o b i n ę .
In the recent ye ars the b i o e l e c t r o c h e m i c a l e x p e r i m e n t s c o n c e r n i n g the b i o l o g i c a l l y a c ti ve m e t a l l o c o m p l e x e s h a v e be e n of great interest. In these e x p e r i m e n t s the iron c o m p l e x e s p l ay the i m p o rta nt role. In ma ny w o r k s [7-29] the o b j e c t s of e x p e r i m e n t s are s y n t h e t i c a lly o b t a i n e d h e m o - s i m i l a r c o m p lex es . One can state, ho wever, lack the e x p e r i m e n t s of Hb o x i d a t i o n to M e t H b on the s o li d e l e c t r o des ęnd on the me rcury. The gr eat Hb m o l e c u l e (mole ab out 65 000) is s u p p o s e d to be an i m p o r t a n t o b s t a c l e in this type of m e a s u r e ments. The e x p e r i m e n t s of the k i n e t i c s of Hb o x i d a t i o n r e a c t i o n on s o li d e l e c t r o d e s w i t h o u t o x i d i z e r s or the i n f l u e n c e of c e r t a i n co m p o u n d s (e.g. m e d i c i n e s ) on the r e a c t i o n rate w o u l d be a p r e c i o u s i n f o r m a t i o n m a i n l y for the i n s t i t u t e s d i r e c t l y c o n n e c t e d with h e a l t h service.
The s t a r t i n g of the a t t e m p t s of e l e c t r o c h e m i c a l r e s e a r c h of Hb s o l u t i o n s has be e n m o t i v a t e d by the i m p o r t a n c e of the pr oblem, by g r ea t i n t e r e s t of the a u t h o r s r e p r e s e n t i n g d i f f e r e n t s c i e n t i f i c b r a n c h e s and by the o b t a i n e d e l e c t r o c h e m i c a l r e s e a r c h po te nti al .
Jx£er^mental
The a t t e m p t s of e l e c t r o c h e m i c a l o x i d a t i o n of Hb s o l u t i o n s we re ca r r i e d out u s in g the c y c l i c v o l t a m m e t r y m e t h o d (CVM) in a s t a n d a r d t h r e e - e l e c t r o d e s y s t e m w i th the gold w o r k i n g e l e c tro de , the c y l i n dric al p l a t i n u m a u x i l i a r y el ec tro de , and the s a t u r a t e d calo mel re f e r e n c e electrode.
The w o r k i n g e l e c t r o d e in fo rm of wire of d i a m e t e r 0 = 1.0 mm and the su rf a c e A = 0.52 c m 2 was p u r i f i e d in the c o n c e n t r a t e d H 2 S 0 4 (p.a.) and e l e c t r o c h e m i c a l l y by m e an s of c y cl ic p o l a r i z a t i o n in the po t e n t i a l ra nge from -1.1 V to +1.4 V with the s w ee p p o l a r i z a t i o n po t e n t i a l rate 0.2 V . s -1 up to the time of o b t a i n i n g the ty pi cal CVM c u rv e in s u p p o r t i n g e l e c t r o l y t e (0.5 M H2S0^).
Eq u i p m e n t set c o n s t r u c t e d in the D e p a r t m e n t of G e n e r a l and In o r g a n i c C h e m i s t r y of the U n i v e r s i t y of Łó dź ( p o t e n t i o s t a t , line ar sw eep p o t e n t i a l g e n e rat or , di gi t a l co nt r o l m e t e r s ) e n a b l e d to o b ta in C V M cur-ves on the B A K- ST register. The s w e e p p o l a r i z a t i o n
a small but cl ear hump that co uld s u g g est Hb o x i d a t i o n pe ak was r e g i s t e r e d .
It has been de ci d e d then to use the o x i d a t e d Hb for the m e a s u rements. For it may be as su med that the o x yg en added to Hb mo le cule, can, in an im portant way, "scireen" hem m o l e c u l e s in cl u d i n g Fe'' and not to let them to the e l e c t r o d e surface.
Mo re over, as the s t r u ctu ra l e x p e r i m e n t s ha ve proved, the oxyg en in Hb m o l e c u l e caus es its d e f o r m a t i o n th ro ugh the protein c h ai ns [32] shift. D e o x i d a t i o n of Hb s o l u t i o n in v a cu um c o n d i t i o n s p r ov ed to be of a small use b e c a use of the long time of de ox ida ti on , stro ng f oaming of the s o l u t i o n and its d i s t i n c t c o n c e n t ra ti on.
Some p o s i t i v e re su lts have been o b t a i n e d duri ng d e o x i d a t i o n th ro ugh the t r a n s m i t t i n g the p u r i f i e d ar gon th ro ugh the solution. Each time argon was t r a n s m i t t e d t h r o ugh the s o l u t i o n in 20 m i n u tes time. Gas flowed out from a c a p i l l a r y (with d i a m e t e r inside 0 = 0.2 mm) under a p r e s s u r e of 50 Atm. The a d d i t i o n of Na BH^ in this te chnics a p pe are d to be needless.
CVM curv es of the d e o x i d i z e d Hb in the ci tr a t e s o l u t i o n were cl ea rly d i f f ere nt from CVM curv es of the s o l u t i o n s w i t h o u t Hb. The s u p p o s e d Hb o x i d a t i o n peak lies too near of the ci tr a t e o x i dati on peak and it is of the shap e of a hump on the rising part of the curve (Figure 2).
Fig. 2. CVM curv es for the 3.8% s o d i u m c i t r ate solution: a) not c o n t ain g Hb; b) cont ain g Hb. Sw eep p o t e n t i a l rate v = 0.02 V . : 1
A mo re cl ear peak ha ve been s u s p e c t e d in the s o l u t i o n s of s o di um c h l o r i d e and s o d i u m fluoride. As 1% NaCl s o l u t i o n c h o s e n as a p h y s i o l o g i c h u mo ur o c c u r i n g in h u m a n o r g a n i s m does not poss es a n t i - c o a g u l a n t pr o p e r t i e s , the m i x t u r e of 1% NaCl and 0. 5% NaF of (different c o m p o s i t i o n has be en us ed as a basi c so lu tio n. In these s o l u t i o n s the p r e s e n c e of Hb al so c a u s e d the f o r m a t i o n of humps on the r i si ng part of the cu rve (F ig ure 3). The p o t e n t i a l at w h ic h h u mps o c c u r e d was h i gh er in c o m p a r i s o n w i th the p o t e n t i a l of the c i t r a t e s o l u t i o n s curves.
S C E
Fig. 3. CV M c u rv es for the s o d i u m c h l o r i d e and s o d i u m f l u o rid e s o l u t i o n (0.5% NaF and 1% NaCl in v o l u m i n a l ratio 100 : 1): a) not c o n t a i n i n g Hb; a) c o n t a i n i n g Hb. Sweep p o t e n t i a l rate v = 0.02 V.s'1
If we a s su me that Hb m o l e c u l e d e v o i d of m o l e c u l a r o x y g e n is able to such d e f o r m a t i o n in the e l e c t r i c field that iron ions F e ++ of this m o l e c u l e c o ul d done an e l e c t r o n t r a n s f e r wi t h the el e c t r o d e , we s h ou ld expe ct a gr eat o v e r v o l t a g e of this process. Thus it is not s t r a n g e that instead of peak p o t e n t i a l eq ual about 0 . 20 0 V (SCE) (near to E° at f e r r i c y a n i d e ti t r a t i o n ) it is found at about 0.750 V (SCE) (in the c i t r a t e so lu t i o n ) or ev en at about 0.95 0 V (SCE) (in NaCl + NaF solutio ns ).
The distinct c h a r a c t e r of the curv es in the ca to d i c part also s u g g e s t s the e l e c t r o d i c o x i d a t i o n of Hb. Peaks a p p e a r i n g on the c a t o d i c part of the curve reffer to the d e s o r p t i o n or r e d u cti on of o x i d a t i o n p r o d uct s of the basic s o l u t i o n (Cl , F ).
In the s o l u tio ns c o n t a i n i n g Hb they occur at d i f f e r e n t p o t e n tials and they have di s t i n c t l y di ff e r e n t c h a r a c t e r (Figure 4).
Fig. 4. CVM curv es for the s o d i u m c h l o rid e and s o di um f l u o rid e solu tion (0.5* NaF and 1% NaCl in vo luminal ratio 1:100): a) not c o n ta in ing Hb; b) c o n t a i n i n g Hb. Sweep p o t e nti al rate v = 0.05 V.s"1
Th ere is ob vi ous ly a p o s s i b i l i t y of the e l e c t r o d i c o x i d a t i o n of the grou ps o c c u rin g both in hem and Hb p r o t e i n chains^. In the pape r [19] de s c r i b i n g the r e d u c t i o n of -S-S- gr oup in p r o t e i n c h ai ns of many s u b s t a n c e s b i o l o g i c a l l y active, it has been stated that Hb in which only -SH grou ps o c cu r does not give any po la r o
g r a p h i c wave in the e x a m i n e d p o t e n t i a l range. H o w e v e r it is little p r o b a b l e that any g r ou p in the p r o t e i n c h ai n w o u l d oxidated mo re e a s i l y than F e + f . On the ba sis of the o b t a i n e d r e s u lts it was a s s u m e d that the o x i d a t i o n of the d e o x i d i z e d Hb is po ss ibl e. The ex p e r i m e n t , however, m e et s a lot of te ch n i c a l troubles.
More d i s t i n c t e f f e c t s are e x p e c t e d from the m e a s u r e m e n t s of the s o l u t i o n s of g r e a t e r Hb c o n c e n t r a t i o n (up to this time im p o s s i b l e b e c a u s e of the s p e c t r o p h o t o m e t e r se ns i t i v i t y ) . The q u a n t i t y i n t e r p r e t a t i o n of the o b t a i n e d Hb o x i d a t i o n hu mps will be p o s s i b l e when the n u m e r i c a l m e t h o d s of e l a b o r a t i n g the re su l t s of CV M m e a s u r e me nt s are used. They e n a b l e to c a l c u l a t e the c h a r a c t e r i s t i c p o in ts of the curve and on this ba sis to e v a l u a t e the k i n e t i c p a r a m e t e r s of e l e c t r o d i c r e a c tio n in a case when the peak of the p r o c e s s is not cl ea r l y s h ap ed (when two p r o c e s s e s take p l ac e at clos e p o t e n tials). This m e t h o d is just be ing test ed on the k n ow n objects.
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B o g u s ł a w G r z e l a k . W i e s ł a w A u g u s ty ni ak , [Jan M ._ Rogoziński! , S t a n i s ł a w Ro m a n o w s k i P R ÓB Y E L E K T R O C H E M I C Z N E G O U T L E N I E N I A H E M O G L O B I N Y KRWI L U D Z K I E J NA E L E K T R O D Z I E ZŁOTEJ M e to dą c h r o n o w o l t a m p e r o m e t r i i c y k l i c z n e j w y k o n a n o prób y u t l e n i e n i a r o z t w o r ó w o d t l e n o w a n e j h e m o g l o b i n y w ró żn y c h a n t y k o a g u l a n - tach na e l e k t r o d z i e złotej. Na p o d s t a w i e o s i ą g n i ę t y c h w y n i k ó w s u g e ruje się, że jest m o ż l i w e u t l e n i e n i e h e m o g l o b i n y w 3,8* r o z t w o r z e c y t r y n i a n u sodu i w m i e s z a n i n a c h 1% NaCl z 0, 5 % N a F .
