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 FOLIA BIOCHIMICA ET BIOPHYS1CA 6. 1988
P aw eł A l e k s a n d e r O s m u l sk i
O X Y G E N A F F I N I T Y
O F C H I R O N O M U S T H UM M I T H UM M I H A E MO G L O B I N S* 4
The insect haemoglobin has been isolated from the fourth larval instar of Chironomus thummi thumrai (Insecta, Diptera). This haemoglo-bin showing high degree of polymorphism was separated into monomeric and dimeric components. Parameters of oxygen affinity of monomeric, dimeric component mixtures and a crude haemoglobins extract were exa-mined. A dithionite - ascorbate solution as an antioxidant system was used during the experiments. High oxygen affinities, an alkaline Bohr effect and any <?o-operativity of the oxygen binding in all the systems investigated were found. These results were discussed in physiological terms as the adaptation of the larvae to their changeable eutrophicated water environment. The mechanism of the action of the antioxidant s y-stem was also proposed.
INTRODUCTION
H a e m o g l o b i n (Hb) is w i d e l y d i s t r i b u t e d o x y g e n c a r r i er p r o te i n
w h i c h o ne c a n f in d in a lm o s t al l v er t eb r at e s. C o n t r a r y to it, its
p r e s e n c e in i n v e r t e b r a t e s is r e s t r i c t e d to o n l y s ome g r o up s or to
s om e r e p r e s e n t a t i v e s of a gro up. H b is a l s o f o un d in s uc h a large
t a x o n o m i c g r o u p as t he I n s ec t a b u t o n l y in a f e w sp ecie s. Am o ng
t h em t hi s of C h i r o n o m u s t h u m mi t h u mm i (Diptera, C h i r o n o m i d a e ) b e
-lo ng s to t he b es t e x a m i n e d so far. T w e n t y y e a r s of i nte n se r e s e
-a r ch w o r k s s h o w e d its h i g h h e t e r o g e n e i t y t w e l ve d i f f e r e n t H b c o
-m p o n e n t s d i s s o l v e d in h a e -m o l y -m p h of a s in g le la r va [2, 14] , its
This work was supported by the Research Program R P.I I.11.1.1.
s tr u c tu r a l o r g an i z a t i o n - five m o n o m e r i c (mol. wt. ca. 15 000) and
se ve n d i m er i c (mol. wt. ca. 32 000) c o mp o ne n ts [10, 14] the kn own
sp at i al s tr u ct u re of the C TT III c om p o ne n t in d i f f e re n t liga nd
s ta te s [11, 17], a nd its f un c t i on a l p ro p e r ti e s [1, 3, 24].
T he da ta i nd ic at e the h ig h o x y g e n aff inity, the p r e se n ce of
an a l k a li n e Bo hr e ff e ct an d the lack of c o - o pe r a t i v i t y of o xy ge n
b i nd i n g in thes e Hb s [2, 8, 9]. Un fo rt un a te l y, the m e n ti o n e d
h ig h o x yg e n a f f in i t y of the Hbs p r o du c es some i mp o rt a nt d i f f i c u
-lties in its c o rr e ct m e as u re m en t s. Th e m a i n p r o b l e m is a m a i n
-te na n ce of the Hbs at the nat ive, f un c ti o na l re du ce state. Th er e
are m a n y wa y s to solve this di f fi c ult y: the u si n g of re d uct an ts
f ol l ow i ng a ge l c hr o ma t og r ap h y, the u si n g c a t al a se or (and) othe r
s pe ci al pr ec au t io n s. In ou r w o r k w e d e c i d e d to us e ot h er re d uc i ng
a ge nt s sy st e ms d i t h i o n i t e - a s c o r b a t e w h i c h m ak e s p o s s i b i l i t y und er
s t r i ct l y d e f i n e d c o n d i t i on s to m e a s u r e all i mp o rt a nt o x y ge n a f
-fi n it y pa ra me ter s. T his p ap e r de al s w i t h the m a k i n g p r og r es s w it h
in v e s t i g at i o n s of thes e p a r a m et e r s in Hb s sh ow i ng the hi g h o xy g en
a f f i n i t y a nd the r es o l vi n g t he w ay s of th is s ys t em action.
MATERIALS AND METHODS
La r va e of C hi r on o m u s t hu m mi t hu mmi we r e c o ll e c t e d in a summe r
p e r i o d (July-Au gust ) as d e s c r i b e d p r e v i o u s l y [14]. O n l y the f o
-u rt h i ns t ar la rva e w e r e t ak e n in to ac count. H b w as is o la t ed us i ng the m e t h o d d e s c r i b e d by W e b e r et al. [24] an d L e y k o an d O s m u l s k i [14]. O x y g e n e q u i l ib r i a of the Hbs we re d e t e r m i n e d us i n g a s pe c t r o p h o t o m e t r i c a l m e t h o d [1] . Th e Hbs s o l u -ti on w as d i s s o l v e d to c o n c e n t r a t i o n 0.2% u s in g 0 . 2 m o l . l ^ p h o s -p h at e or T r i s - H C l buffer. A b s o r b a n c e m e a s u r e m e n t s w e r e m a d e w it h a S p e c o r d U V - V I S or a S p e c t r o p h o t o m e t e r V S U - 2 G ( C ar l- Ze is s Jena) at 560 a nd 575 nm. E q u i l i b r a t i o n t ime s af te r e a ch a d d i t i o n of air p o r t i o n w e r e 5 min. D e o x y g e n a t i o n of th e H b s o l u t io n w a s p e r -f o rm e d b y p a s s i n g a rg o n t h r o u g h a t on o me t er for ab o ut o ne hour. T h e n 0.1 m l of s o di u m d i t h i o n i t e s o l u ti o n of a pp r o p r ia t e c o n c e n
-t r a -t i o n w as d r o p p e d in-to the m e a s u r i n g ch amber. Next, 0.1 m l of
a s c o rb i c a c id s o l u t i o n w a s dr oppe d. A ft e r e a c h a d d i t io n of the
r e d u ct a n t s o l u t i o n ar g on w a s p a s s e d t h r o u g h a t o n o me t er for abou t
15 min. Th e c o n c e n t r a t i o n of s o d i u m d i t h i o n i t e a nd a s c or b i c aci d
in the H b s am pl e w e r e 3.85 m g % a n d 7.70 m g% r es p ec ti ve ly . The
RESULTS O ur r e su l t s of o x y g e n a f f i n i t y m e a s u r m e n t s are sh ow n 1-4 a nd ar e s u m m a r i z e d in Tab. 1 w h e r e the d a ta of ot he r 450 500 600 700 in Fi gs au th or s nm 800 __L-24 2 2 20 18 16 14 » 1000 c '1
Fig. 1 ‘ Absorption spectra of a mixture of the monomeric components (---- ) an oxy form spectrum in the presence of the reducing system; ( — — ) 1 - MetHb, 2 - the same after ascorbate addition (5 min), 3-7 - the same alter
dithionite addition (2, 5, 7, 10, 15 min.)
Rys. 1. Widma absorpcyjne mieszaniny składników monomerycznych (---- ) widmo formy utlenowanej w obecności układu redukującego, ( ) 1 MetHb, 2 - po dodaniu askorbinianu (5 rain), 3-7 - po dodaniu podsiarczynu
w 2 0 4 0 6 0 8 0 1 0 0 p O j [ m m H g ] • M b m o n H b d i m H b o o ł k p H F i g . 2 . O x y g e n e q u i l i b r i u m c u r v e s o f t h e m i x -t u r e s o f t h e i n v e s t i g a t e d H b s „ , , . , ., . _ , Ł F i g . 3 . O x y g e n a t i o n c h a r a c t e r i s t i c o f t h e m i x t u r e o f t h e t o t a l H b s R y s . 2 . K r z y w e d y s o c j a c j i t l e n o w e j m i e s z a n i n „ „ . . . _ . . „ b a d a n y c h h e m o g l o b i n R y s - 3 ‘ Z a l e z n o s c P 5 0 i p a r a m e t r u n H b c a ł k o w i t e j o d p H
a)
b)
Fig. 4. Oxygenation characteristic of the mixtures of monomeric and dimeric Hbs a) the pH dependence of the n parameter, b) the pH dependence of the p^Q pa
rameter
Rys. 4. Charakterystyka utlenowania mieszaniny Hb monomerycznych i mieszaniny Hb dimerycznych
T a b l e 1 Parameters of the oxygen affinities
of the C. thummi thummi haemoglobins Parametry powinowactwa tlenowego hemoglobin
larw C. thummi thiunmi
The kind of the system (mm Hg, pH 7)P50 Bohr effect -Alogp5Q/ApH n (at pH 7) References Mixture of 0.27 n.d. 1.15 [22] all the Hb 0.5 n.d. 1.0 ['7]
components 1.67 t 0.37 0.70 ± 0.13 1.10 ± 0.07 this work
Mixture of 1.04 ± 0.09 0.80 ± 0.21 0.95 ± 0.19 this work
the monomeric 1.27a 0.0 1.0 [24]
components 0.63b 0.30 1.0 [24]
Mixture of 1.57 1.55 1.0 [ 9]
the dimeric 1.76 ± 0.19 1.29 ± 0.22 1.13 ± 0.23 this work
components 1.50° 0.79 1.0 [24]
a The component CTT I (monomeric). b The component CTT III (monomeric). c The component CTT VI (dimeric). N o t e : n.d. - not determined.
are a l s o g i ve n for the c om pa ris on . O u r f in d in g s a gr e e w e l l w i t h
th os e of W e b e r et al. [24] or G e r s o n d e e t al. [8,9]
w i t h i n all t hr ee points: the h i g h o x yg e n aff init y, the p re s en c e
of the a l k a l i ne B oh r e f fe c t a nd the la ck of the c o - o p e r a t i v i t y
of o x y g e n binding. S in ce o ur r e d uc i ng s y s t em w as n ot r em ov e d f ro m t he H b s o l u -t io n i-t w as n e c e s s a r y to ta ke i nt o a c co u nt the i n fl u en c e of this s y st e m on o x y g e n a f f i n it y p ar a me te rs . T he s im p le l ine ar d e p e n -d en c e of r e -d u ct a n t s c o n c e n t r a t i o n in th e ra n ge 3-24 mg % on log p 5Q an d lo g K w a s found, so a ft er a l ine ar i n t e rp o l a t i o n of g iv en v al u e s to the c o n c e n t r a t i o n of th e s ys t em e qu a l to 0 o ne c a n c o r -re ct r esults.
D u r i n g all the time of the e x p e r i me n t r un th e d é n a t u r a t i o n of
the H b w a s n o t o b s e r v e d a l t h o u g h s o me t im e s the fu ll o x y ge n l o
-a d in g b y the H b w as not p o s s i b l e (the a/0 b an d s ra ti o d e c r e a se d
fi-n i s h i fi-n g w a s le ss t ha fi-n 10%. T he sh a pe of the o x y g e n d i s s oc i a t i o n
c u r v es w a s h y p e r b o l i c (Fig. 2) s h o w in g n o n - c o o p e r a t i v i t y in the
o x y g e n b i n d i n g so v a lu e s of a "h" p a r a m e t e r w e r e in a ra ng e 0.95
-1. 13 (Figs 3 an d 4). A n e x t r e m e l y h i gh o x y g e n a f f i n i t y w as
fo u nd for m i x t u r e of t he m o n o m e r i c Hbs, w ha t is m o r e , for this
m o n o m e r i c Hb s m i x t u r e the h e t e r o t r o p i c a l l o s t e r i s m w as fo und - th e B o h r e f f e c t w as (-filog p5Q /ApH) eq u al 0.80. DISCUSSION It is n e c e s s a r y to t ak e i nt o a c c ou n t t h r e e .i m po r ta n t p r o b le m s c o n n e c t e d w i t h o ur re sults: 1) the o x y g e n a f f i n i t y of the C. t h um m i t h um m i H bs in t he r e s -p e c t of t he m o d e of t he l arv ae life; 2) th e c o m p a r i s o n of ou r r e su l t s w i t h t ho s e g i v e n b y ot he r a u th o r s w i t h r e g a r d t o the u s e f u l n e s s of ou r r e d u c i n g system; 3) t he m e c h a n i s m of the a c t i o n of t he r e d u c t a n t s s y s te m d u -r in g the o x y g e n a f f i n i t y m ea s u -r e m e nt s . T h e p h y s i o l o g i c a l s i g n i f i c a n c e of the C. t h u m mi t h um m i Hb s in t he l igh t of t h ei r f u n c t i o n al p ro p er t ie s . T he hy pox ic , b e n t hi c h a -b i t a t of C. t h um m i t h um m i l a rv a e is w e ll c o n n e c t e d w i t h t he f oun d h i g h o x y g e n a f f i n i t i e s of t h e ir Hbs. O n th is g r o u n d o ne c a n s u g -g e s t th e r ol e of t h es e p r o t e i n s as a n o x y g e n store. T h i s s imp le p i c t u r e d o es n ot i n c l ud e t h re e i m p o r t a nt facts: a c i r c u l a t i o n of the h ae m o l y m ph , c h a n g e s of H bs c o n c e n t r a t i o n in t he r e sp e ct of w a t e r a e r a t i o n c o n d i t i o n s a n d the p r e s e n c e of t he a l k al i ne Bo hr e ffec t. T h e c i r c u l a t i o n of the h a e m o l y m p h w i t h t he f r e el y d i s -s o lv e d H b-s t h r o u g h th e c o m p a r t m e n t s of the l ar va l body, t ho u gh n ot s p e c i a l l y e f f e c t i v e m a k e s p o s s i b l e the d i r e c t i o n a l o x yg e n t r a n s p o r t [24]. T h e c o n c e n t r a t i o n of the t ot a l H b i nc r ea s es d u -r in g t he l a-r v al d e v e l o p m e n t a nd h y p o x i a p e r i o d s in w at e r [7, 14, 19-21] . Ag ain , th e d i m e r i c / m o n o m e r i c r at i o of Hb s c h an g es in a r e s p e c t to e n v i r o n m e n t a l c o n d i t i o n s so the b e st s u pp l y of o x yg e n is m a i n t a i n e d [14]. T h e a l k a l i n e B oh r e f f e c t h as t wo as pects: a s it e s p e c i fi c a n d a t im e s p e c i f i c [24]. T h e f ir st v i e w is a d i r e c t r e p e r c u s s i o n of the a bo ve m e n t i o -n e d c o m p a r t m e -n t a t i o -n of t he l ar v ae a n d c o n n e c t e d w i t h it i nt e rn a l pH d i f f er e n t i a t i o n . T h e s e c o n d is r e l at e d to a p e r i o d i c v e n t i l a -t o r y b e h a v i o u r of -t he l ar va e [19-21, 23]. T he o x y g e n l o ad i ng o c
-cur s d u ri n g the v e n t i la t i o n p e ri o d fo l lo w ed by the int en se o x y
-ge n co n su mp tio n. W it h o u t the al ka l in e Bo hr e ff e ct , th e ox y ge n u n
-l o ad i ng w o u -l d h a pp e n o n l y at its e x t r e m el y low t e ns i on in the
t is su es [22, 24]. S u m m a r iz i n g all thes e f in din gs and c o nc e p ti o ns
we c a n so lv e the o ld q u e s t i o n of the fu n ct i on of th es e Hbs: an
o xy g en st ore or a t r an s p o rt e r? In the light of ou r pr es e nt k n o w
-ledge we ca n p r op o se the f ol l ow i n g ac t ion of the Hbs system: it
f a ci l it a te s ox y g e n d if fu si on, t r an s po r ts it t hr o u g h the b od y s e
-gments, a cc u m u l at e s o xy g e n d u r i n g the v e nt i l a t i o n a c t i v i t y and
r el e a se s it in the ti ss ue s of h ig h m e t a b o li c a c t i v it y and d ur i ng
the re st periods.
T he o x yg e n a f f i n i t y d e t e r m i n a t i o n in the p r e se n ce of the r e d u
-c i ng system. Ou r re su lts are c o n s i s te n t w i th thos e p u b li s h e d p r e
-v i o u s l y by othe r authors, but g e n er a l l y sho w the lower o xy g en a f
-fi n it i es of C. t hu mmi t hu mmi Hb s [1, 8, 9]. Th is m a y be c o n n ec t ed
w i th the fact tha t som e a u th o rs d id not ta ke into ac co u nt the h igh
a u to x i d a t i o n rat e of the se pr ot eins . O th e r da n ge r w h i c h c an i n
fl ue n ce on the e r r o ne o u s l y h ig h v al u es is an in co m pl e te d e o x y g e n a -t io n of a sample.
Ou r s y st e m h av e m a n y p o s s i ti v e p rop er ti es : 1) it do es not
c r ea t e d e t e c t a b l e am ou n ts of h em o - a nd h e m i c h r o m o g e n e s ; 2) it d o
-es not c h an g e the p H v al u e of a m e d i u m (in the ra ng e ±0.05 of pH
unit ); 3) it e x c lu d es the d i f fi c u l t to d ef i ne i n t er a c ti o ns of
the Hb (or Mb) w i t h ot h er e v e n t u a l l y ad d ed p r o te i ns (e.g. cata-
lase) an d 4) the s t a b il i t y of the re d uc i n g s ys t em in the pH r a
-ng e 6 - 9 m ak e s p o ss i bl e the Bo h r ef f ec t study. Th i s sy s t e m has
al so th e n e ga t iv e pr o p e rt i e s of course: 1) it ne ed s c a l c ul a t i o n
of the s pe ci a l c o r r e c t i o n for the p re s e nc e of the s y s te m in the
i n v e s ti g a t e d solu tion ; 2) it m a k e s p o s si b i l i t y of the o x yg e n c o n
-s u mp t i o n b y the -sy -s t e m un d e r s p ec i fi c ex p e r im e n t a l c o n di t i o n s so
lowe r o xy g e n a f fi n it i es m a y be rece ived .
R e a c t i o n of the r e d uc i ng s ys t e m w i t h C. th u mmi thumm i H bs . T h e
-re a-re th -ree as pe ct s of the r e a ct i o n of the r ed u ci n g s ys t e m w i t h
C. th um mi th u mm i Hbs i.e. a r ea c t i o n w i t h d i t h io n i t e anion, wi t h
a s c o rb a t e an i on an d a d i t h i o n i t e a s c o rb a te in te rac tion . Th e m e
-c h a n i s m of the se re a ct i on s in the a b se n ce of ox y g e n is m or e s i m
-pl e an d b et t er k no w n th an th o se in its p r e se n c e [18, 25]. Or ga n ic
r e d u c i n g a ge nt s c an r ed uc e an o x i d i z e d f or m of a h a e m p r o te i n in
th r ee f u n d a m e nt a l ways: an e l e c t r o n t r a ns f er to the h a e m t hro u gh
o-n it e io o-n c ao-n r ea ct in thi s m a n n e r [5]), t h r ou g h the p er i p h e ri c a l
gr o u p s of the p o r p h y r i n or t h r o u g h the gl o b i n (the p r op o s e d w a y of
the a s c o r b a t e io n r e a c t i o n [18]).
t ha t s i n ce the a s c o r b a t e a n i on is t oo lar ge to e n t er i nto the h ae m
p o c k e t it m u s t e xi s t t he g l o b i n - w a y of the e l e c t r o n tra nsfe r. T he n
t he " s t re n gt h " of the r e d u c i ng p r o p e r t i e s of a s c o r b a te m a y d e p e nd on a p o s i t i o n a nd a k i n d of a mi n o a ci d r e s id u es of the g l o b i n , p a r -t i c u l a r l y -t r y p -t o p h a n or a r g i n i n e r es idu es. In f a c t, t he s e s u g g e -s t io n -s w e r e f o u nd c o n f i r m a t i o n o n the b as i s of k i n et i c s tu di e s on the r e d u c t i o n of i m i d a z o l m e t m y o g l o b i n b y a s c o r b ic a c i d [18 a nd r e -f e r e nc e s c i t e d t h e r e i n ] . T h e r e a c t i o n of M e t H b w i t h a sc o r b at e l e -ad s to r e d u c e d H b a nd a s c o r b a t e ra dica l. F r om tw o a s c o rb a te r a d i -c a ls a s -c o r b a t e a n i o n a nd d e h y d r o a s c o r b a t e is formed. Th e m e c h a n i s m of th e a n a er o b i c r e d u c t i o n of the h a e m p r o t e i n s by d i t hi o n i t e is d e s c r i b e d b y s 2 ° 4 _ 5 = 5 2 S 0 2 SOj + M e t H b ( H20) --- *■ SO-jH" + H + + Hb so t he e f f e c t i v e r e d u c i n g .agent is a s ul f ur d i o x i d e r a di ca l [25]. A f t e r a n o x y g e n a d d i t i o n o ur s y s t e m b e c o me s m o r e c o m p l i c a t e d an d w h a t is m o r e no t c l e a r l y d ef in ed . W e c an s u pp o se on the ba si s of a v a i l a b l e l i t e r a t u r e th e f o l l o w i n g s ch e me [4, 5, 6, 12, 15]. As an a ct i ve i n t e rm e d i a t e s u p er o x i d e r a d ic a l is p r o d u c e d in
t he a b ov e p r oce ss . H y d r o g e n p e r o x i d e r e ac t s t he n w i t h f err ic and
f e rr o us i on of the h a e m w h a t g iv e s h y d r o x y l r a di c a l an d f er ryl d e -r i v a t i v e of h a em o g l o b i n. T he -r e a c t i o n of f e-r -ry l H b w i t h H b a n d HbOj le ads to fe rr i Hb g e n e r a t i o n [26]. In th e p r e s e n c e of o x y g e n the r a d i c a l a n i o n m o n o m er , SO^ m a y c r e a t e h y d r o g e n p e r o x i d e t h r o u g h a s u -p e r o x i d e d i s m u t a t i o n [25]. T h us M e t H b w i l l be f o r me d if o n l y the t h i o n i t e a n d a s c o r b a t e m a k e s p o s s i b l e the i n t e r a c t i o n of sulf ur d i o x i d e r a di c al w i t h a n a s c o r b a t e r a d ic a l or w i t h o th er i n t e r m e -di ates. T h e n ferryl d e r i v a t i v e of H b is n ot o b s e r v e d an d a c on tr ib
u-tion of the f er r ic form is m in i ma l . Unfortunately, d e t ai l s of
th es e b l o c k i n g r e a c t i o n s ar e n ot c le ar for us. W e t h i n k th at th es e
T s u k a h a r a an d Y a m a m o t o [18] r ep or te d
AH
H.+
free r adi c al r ea c ti o ns m a y be c o n n e ct e d w it h the e as e of the C.
th um mi t hum mi h a e m o g lo b i n s o x id a t i o n [14, 16]. Now, we tr y to
cl ear up the re ac t io n of a s c or b ic aci d w i t h d i ff e r e n t h a e m p r o
-tei ns (work in pr og ress ). T h e r e is st il l m u c h to be le arn t b oth
ab out th e w e l l kn o w n r e a ct i o n of d i t h i on i t e an i on as a red uc tor
an d abo ut not so w el l u n d e r s t o o d re a ct i o n of asco rbate.
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Chair of Biophysics University of Łódź
Paweł Aleksander Osmulski *
POWINOWACTWO TLENOWE HEMOGLOBIN LARW CHIRONOMUS THUMMI THUMMI
Z larw czwartego stadi-um Chironomus thummi thummi wyizolowano białko odde-chowe o właściwościach hemoglobiny. Białko to wykazuje wysoki stopień niejedno-rodności, gdyż metodami elektroforetycznymi i chromatografii jonowymiennej stwierdzono obecność 12 różnych składników w hemolimfie pojedynczej larwy. Z ba-dano parametry powinowactwa tlenowego mieszaniny składników monomerycznych, di- merycznych, a także mieszaniny wszystkich składników. Podczas wykonywania po -miarów zastosowano układ redukujący podsiarczyn sodu - kwas askorbinowy, ze wzglądu na wysoką stałą szybkości samoutleniania badanych hemoglobin. We wszy stkich zbadanych układach stwierdzono wysokie powinowactwo tlenowe, obecność alkalicznego efektu Bohra i brak kooperatywności wiązania tlenu. Wyniki te zo-stały przeanalizowane pod kątem fizjologicznego przystosowywania się larw do silnie zeutrofizowanego środowiska wodnego o zmiennych właściwościach tleno-wych. Zaproponowano również mechanizm działania układu redukującego w warun kach tlenowych i beztlenowych.
