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34 Radiation Chemical Studies on Oxygen-Carrying Proteins: Hemocyanin

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JACK SCHUBERT, E. R. WHITE, and L. F. BECKER, JR. Radiation Health Division, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pa. 15213

The oxygen-carrying capacity of the nonheme copper pro­ tein, hemocyanin, is reduced by irradiation of the oxygenated protein with gamma and x-rays with G(-O ) = 0.9-1.2. Measurements are made on hemocyanin in its own serum at relatively low doses (0-80 krads). Oxygenated hemo­ cyanin is resistant to irradiation but loses its oxygen-carrying capacity only when deoxygenated following irradiation in the oxygenated state. Irradiation of the deoxygenated form is without effect on the oxygen-carrying capacity. The effect of irradiation on hemocyanin involves the oxidation of protein-bound copper atoms to the Cu(II) state by radiolytic hydrogen peroxide in the same manner as is produced by adding H O to unirradiated hemocyanins. The effects of organic peroxides, pH, urea, catalase, and other factors on the oxygenation of hemocyanin are described. 2

2

2

/~\ne of the most active areas of research i n b i o l o g i c a l systems is c o n c e r n e d w i t h t h e m e c h a n i s m of a c t i v a t i o n a n d u t i l i z a t i o n of o x y g e n (15).

O x y g e n p l a y s a n i m p o r t a n t a n d s p e c i a l role w i t h respect to r a d i a ­

t i o n d a m a g e a n d p r o t e c t i o n i n b o t h r a d i o b i o l o g y (2, 39) a n d r a d i a t i o n c h e m i s t r y (5, 43).

A s a m o d e l r a d i o b i o l o g i c a l system w e h a v e

chosen

to investigate t h e effects of i o n i z i n g r a d i a t i o n o n the o x y g e n a t i o n reac­ tions of o x y g e n - c a r r y i n g proteins u n d e r as n a t u r a l c o n d i t i o n s as possible. Since w e are s t u d y i n g t h e effects of r a d i a t i o n o n t h e f u n c t i o n a l b e h a v i o r of proteins, w e g e n e r a l l y use r e l a t i v e l y s m a l l total r a d i a t i o n doses ( 0 - 0 . 1 M r a d ) rather t h a n a c o m b i n a t i o n of h i g h r a d i a t i o n doses (^—0.5-5 M r a d s ) and unphysiological conditions w h i c h produce

gross

damage,

q u e s t i o n a b l e relevance to b i o l o g i c a l systems. 480 Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

having

34.

SCHUBERT E T A L .

481

Hemocyanin

I n this report w e present f u r t h e r results of o u r investigations (34, 37) on the action of gamma-irradiation o n the nonheme copper protein, hemocyanin.

oxygen-carrying

T h i s p r o t e i n serves as a n e s p e c i a l l y c o n ­

v e n i e n t r a d i o b i o l o g i c a l m o d e l because its o x y g e n a t i o n reactions c a n b e s t u d i e d o n t h e p r o t e i n as i t exists n a t u r a l l y i n its o w n s e r u m , w i t h o u t i s o l a t i o n or d i s r u p t i v e p u r i f i c a t i o n procedures.

T h e use of a

copper

p r o t e i n is of p a r t i c u l a r interest i n v i e w of suggestions that c o p p e r pos­ sesses some u n i q u e characteristics 36).

a n d roles i n r a d i o b i o l o g y ( J , 19, 35,

W e h a v e a l r e a d y s h o w n that t h e o x y g e n - c a r r y i n g c a p a c i t y of t w o

types of h e m o c y a n i n as m e a s u r e d b y o p t i c a l a b s o r p t i o n at 340 m/x d e ­

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creases w i t h i n c r e a s i n g r a d i a t i o n dose G ( - 0 ) of about 1.0 (34, 2

giving

initial yield

values

of

37).

P r e v i o u s investigations

(4, 32, 41, 42)

o n t h e actions

r a d i a t i o n o n h e m o c y a n i n i n v o l v e d doses of 2 χ

of i o n i z i n g

1 0 to 6 Χ 1 0 rads a n d 5

5

h i g h e r . S v e d b e r g (41, 42) s h o w e d that i r r a d i a t i o n w i t h α-particles s p l i t h e m o c y a n i n f r o m t h e s n a i l Helix

i n t o halves a n d e v e n t u a l l y

pomatia

eighths. P r e s u m a b l y h y d r o g e n b o n d s w e r e b r o k e n b y d i r e c t a c t i o n of t h e h e a v y i o n i z i n g tracks.

C h e m i c a l changes m u s t h a v e o c c u r r e d because

the fragments c o u l d not b e reconstituted. w h e n h e m o c y a n i n f r o m Limulus (32).

B a r r o n (4)

A s i m i l a r s p l i t t i n g takes p l a c e

polyphemus

is i r r a d i a t e d w i t h

d e m o n s t r a t e d that large doses ( 4 - 6 X

x-rays

1 0 r a d s ) of 5

x - i r r a d i a t i o n of h e m o c y a n i n i n d i l u t e solutions c a u s e d t h e c o p p e r - p r o t e i n b o n d to r u p t u r e .

Properties of

Hemocyanins

T h e f o u r m a i n groups of o x y g e n - c a r r y i n g p r o t e i n s — t h e h e m o g l o b i n s , c h l o r o c r u o r i n s , h e m e r y t h r i n s , hemocyanins—possess

t h e v i t a l f u n c t i o n of

transport a n d storage of o x y g e n i n t h e a n i m a l k i n g d o m ( 1 5 , 23).

They

react r e v e r s i b l y w i t h m o l e c u l a r o x y g e n at r e l a t i v e l y h i g h p a r t i a l pressures a n d release i t t o tissues w h e r e t h e p a r t i a l pressure is l o w . H e m o g l o b i n s a n d c h l o r o c r u o r i n s possess a n i r o n - c o n t a i n i n g prosthetic

group a n d a

p r o t e i n . T h e o x y g e n b i n d i n g site resides w i t h t h e i r o n a t o m centered i n the p o r p h y r i n r i n g w h i l e t h e g l o b i n makes t h e prosthetic g r o u p soluble a n d its reactions w i t h o x y g e n reversible. I n b o t h h e m o g l o b i n a n d c h l o r o c r u o r i n t h e m o l e c u l a r o x y g e n c o m b i n e s i n a 1:1 ratio w i t h t h e F e a t o m . H e m e r y t h r i n , w h i c h contains i r o n , a n d h e m o c y a n i n , w h i c h contains c o p p e r , are n o n h e m e o x y g e n - c a r r y i n g proteins i n w h i c h t h e m e t a l appears to b e b o u n d d i r e c t l y to the p r o t e i n via t h e f u n c t i o n a l g r o u p i n g s of o n e or m o r e a m i n o a c i d residues.

T h e m e t a l to o x y g e n ratio appears to b e

2:1 i n b o t h h e m e r y t h r i n a n d h e m o c y a n i n i n contrast to the 1:1 ratio f o u n d i n t h e h e m e proteins (13, 23, 26).

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

482

RADIATION CHEMISTRY

1

T h e existence of other n a t u r a l o c c u r r i n g o x y g e n carriers has not b e e n p r o v e d . P r e s u m p t i v e e v i d e n c e for a n o x y g e n - c a r r y i n g f u n c t i o n has b e e n a s c r i b e d to a v a n a d i u m - c o n t a i n i n g p r o t e i n , h e m o v a n a d i u m (16)

found

i n the m a r i n e organisms of the t u n i c a t e f a m i l y . H e m o c y a n i n s f r o m v a r y i n g sources c o p p e r , 0.8 to 1 . 2 %

c o n t a i n f r o m 0.15

to

0.26%

s u l f u r , a n d m o l e c u l a r w e i g h t s w h i c h v a r y consider­

a b l y because of the a g g r e g a t i o n of smaller units ( 13, 33 ). T h e a m i n o a c i d c o m p o s i t i o n of h e m o c y a n i n s f r o m t e n different species h a v e b e e n deter­ m i n e d (14).

R e m o v a l of c o p p e r or o x y g e n does not m o d i f y the p r o t e i n

c o m p o s i t i o n or p r o d u c e a n y m e a s u r a b l e change as m e a s u r e d b y o p t i c a l

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r o t a t o r y d i s p e r s i o n o n the c o n f o r m a t i o n of the p r o t e i n structure

(7).

T h e state of a g g r e g a t i o n of the subunits determines, i n part, the o x y g e n a ­ t i o n properties of the p r o t e i n (17,

18).

T h e proteins are colorless w h e n

d e o x y g e n a t e d a n d d e e p b l u e w h e n oxygenated.

T h e oxygenated protein

is r e a d i l y d e o x y g e n a t e d b y passage of a stream of oxygen-free h e l i u m t h r o u g h a s o l u t i o n of the p r o t e i n . T h e p r o t e i n - b o u n d c o p p e r c a n be r e m o v e d b y d i a l y s i s against c y a ­ n i d e , sulfide, a n d other c o p p e r c o m p l e x i n g agents.

Subsequent reintro­

d u c t i o n of the c o p p e r restores the o x y g e n a t i n g a b i l i t y of h e m o c y a n i n . H o w e v e r , the resynthesis of h e m o c y a n i n f r o m a p o h e m o c y a n i n c a n a c h i e v e d o n l y w i t h c u p r o u s , not c u p r i c salts (6, 13).

be

Practically quanti­

tative r e c o n s t i t u t i o n is o b t a i n e d b y the use of the a c e t o n i t r i l e c o m p l e x of c o p p e r (21).

T h e a p o p r o t e i n appears specific f o r c o p p e r since

b i n d i n g of other m e t a l ions denatures the p r o t e i n M a n y investigations h a v e

been

made

the

(6).

to i d e n t i f y the

ligands i n

h e m o c y a n i n responsible for the b i n d i n g of c o p p e r a n d the o x i d a t i o n state of the c o p p e r itself (6, solved.

9, 13, 23).

T h e s e questions h a v e not b e e n re­

M o r e recently, h o w e v e r , measurements

of the o p t i c a l rotatory

dispersion ( O D ) and circular dichroism ( C D ) on hemocyanin obtained f r o m Octopus

vulgaris

a n d Loligo

pealei h a v e p r o d u c e d c o n v i n c i n g e v i ­

dence, a l b e i t c i r c u m s t a n t i a l , o n the b i n d i n g of c o p p e r to h e m o c y a n i n ( 7, 45, 46).

V a n H o l d e (46)

c o m p a r e d the C D spectra o b t a i n e d for the

m o l l u s c a n h e m o c y a n i n w i t h those of p e p t i d e - C u ( I I ) complexes.

Only a

h i s t i d i n e - c o n t a i n i n g c o m p l e x s h o w e d C D spectra r e s e m b l i n g those f o u n d f o r the h e m o c y a n i n s . T h u s , as has b e e n i n f e r r e d f r o m other observations, the h i s t i d y l residues are at least p a r t i a l l y responsible for the b i n d i n g of copper i n hemocyanins. T h e investigations of F e l s e n f e l d (10)

o n the a c t i o n of h y d r o g e n

p e r o x i d e o n h e m o c y a n i n has p r o v i d e d interesting i n f o r m a t i o n w h i c h , i n fact, explains the r a d i a t i o n effects o b s e r v e d thus far. H e f o u n d that the d e o x y g e n a t e d h e m o c y a n i n w a s m u c h m o r e sensitive to attack t h a n the o x y g e n a t e d h e m o c y a n i n as m e a s u r e d b y the o x y g e n - c a r r y i n g c a p a c i t y . T h e p e r o x i d e appears to act b y o x i d i z i n g the C u ( I )

of

deoxygenated

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

34.

SCHUBERT E T A L .

h e m o c y a n i n to the C u ( I I ) Busycotypus

483

Hemocyanin

state.

T h e hemocyanin from the mollusk

c a n b e regenerated b y t h e use of r e d u c i n g

canalicuhtum

agents a n d b y t h e use of excess p e r o x i d e ( 1 0 ) . T h e p r o d u c t of t h e per­ oxide o x i d a t i o n was d e s i g n a t e d as m e t h e m o c y a n i n i n a n a l o g y to m e t h e m o g l o b i n since i n b o t h cases specific o x i d a t i o n of the m e t a l i o n o c c u r r e d w i t h a c o n c o m i t a n t loss of p h y s i o l o g i c a l a c t i v i t y . H e m o c y a n i n possesses a catalase-like a c t i o n (10, 12), albeit a w e a k one, i n that i t decomposes h y d r o g e n p e r o x i d e i n t o w a t e r a n d o x y g e n just as t h e h e m e p r o t e i n catalase.

T h e catalase-like a c t i o n of h e m o c y a n i n is

d u e to the p r o t e i n - b o u n d c o p p e r since neither copper-free h e m o c y a n i n Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch034

nor

free

copper

ions

exhibit

activity

under

equivalent

conditions.

C o p p e r - a m i n o a c i d complexes h a v e b e e n tested f o r catalase-like a c t i o n , a n d it w a s r e p o r t e d that o n l y t h e c o p p e r - a r g i n i n e c o m p l e x possesses catalase-like a c t i v i t y (13).

H o w e v e r , w e h a v e f o u n d (37)

as h a v e other

investigators (29, 40), that other a m i n o a c i d complexes of c o p p e r also decompose H 0 . 2

2

O x y g e n a t e d h e m o c y a n i n exhibits a b s o r p t i o n b a n d s i n t h e v i s i b l e a n d u l t r a v i o l e t regions at about 278, 345, a n d near 600 πΐμ. T h e b a n d at 278 m μ is c o m m o n to a l l proteins w h i l e t h e other t w o , c a l l e d c o p p e r b a n d s , d i s a p p e a r u p o n d e o x y g e n a t i o n or r e m o v a l of the c o p p e r (13).

Neither

o x y g e n a t e d or d e o x y g e n a t e d h e m o c y a n i n give a n E P R s i g n a l except w h e n the p r o t e i n is d e n a t u r e d at w h i c h p o i n t t h e c o p p e r becomes p a r a m a g ­ netic (27).

H o w e v e r , w h e n d e n a t u r e d h e m o c y a n i n is regenerated

cysteine o r H 0 , t h e E P R s i g n a l disappears 2

2

with

(22).

T h e o x i d a t i o n state of t h e c o p p e r i n h e m o c y a n i n s has b e e n t h e sub­ ject of m u c h i n v e s t i g a t i o n a n d d i s c u s s i o n (11, 23, 24, 25, 27, 30). V a n H o l d e (46)

has r e v i e w e d t h e e v i d e n c e r e g a r d i n g t h e o x i d a t i o n state of

c o p p e r i n c o p p e r proteins. H e points o u t that t h e b o n d at a b o u t 340 m/x is v e r y strong i n t h e h e m o c y a n i n s a n d corresponds to t h e i r a b i l i t y to b i n d o x y g e n a n d to t h e presence of c u p r i c c o p p e r .

T h e c o n c l u s i o n is d r a w n

that i n o x y h e m o c y a n i n the c o p p e r is at least p a r t i a l l y i n the C u ( I I ) f o r m a n d that t h e absence of a n E P R s i g n a l indicates either

electron

p a i r i n g w i t h t h e o x y g e n or b e t w e e n t h e c u p r i c ions. A tentative m e c h a ­ nism for 0

2

b i n d i n g i n h e m o c y a n i n s is presented

(46)

i n w h i c h it is

a s s u m e d that i n d e o x y h e m o c y a n i n o n e c o o r d i n a t i o n site o n each c u p r o u s i o n is o c c u p i e d b y a w a t e r m o l e c u l e . W h e n these are r e p l a c e d b y 0 , 2

d i s t o r t i o n of t h e c o o r d i n a t i o n is r e q u i r e d because of t h e smaller size of 0 . 2

E l e c t r o n transfer to 0

2

is f a c i l i t a t e d b y t h e longer b o n d l e n g t h i n

the 0 ~ i o n . 2

2

Experimental Limulus ( t h e horseshoe or k i n g c r a b ) a n d Busycotypus (Busycon, the c h a n n e l e d w h e l k ) h e m o c y a n i n h e m o l y m p h are o b t a i n e d f r o m t h e

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

484

RADIATION CHEMISTRY

1

M a r i n e B i o l o g i c a l L a b o r a t o r y , W o o d s H o l e , M a s s . T h e h e m o l y m p h is s h i p p e d c o l d i n i n s u l a t e d containers via air the same d a y the animals are b l e d . T h e h e m o c y a n i n is " c o n d i t i o n e d " u p o n a r r i v a l . T h i s consists of r e m o v a l of large c l u m p s of c l o t t e d m a t e r i a l w i t h a glass r o d . T h e s o l u t i o n is t h e n filtered ( R e e v e A n g e l flutted filter p a p e r # 8 0 2 ) . S u b s e q u e n t l y , the s a m p l e is o x y g e n a t e d b y b u b b l i n g o x y g e n t h r o u g h the s o l u t i o n for a b o u t t e n m i n u t e s at a rate of 20 m l . p e r m i n u t e . T h e o x y g e n a t e d h e m o ­ l y m p h is p l a c e d i n a 350 m l . gas w a s h i n g bottle w i t h inlet a n d f r i t t e d d i s c at the b o t t o m . T h e bottle is s u r r o u n d e d b y ice, a n d a d e o x y g e n a t i o n c y c l e is c a r r i e d out b y b u b b l i n g especially p u r i f i e d h e l i u m gas ( 0 < 0.001% b y v o l u m e ) at a rate of 40 m l . per m i n u t e u n t i l the s o l u t i o n is colorless. T w o a d d i t i o n a l r e o x y g e n a t i o n a n d d e o x y g e n a t i o n cycles are c a r r i e d out. A f t e r the final o x y g e n a t i o n the s o l u t i o n is filtered, a f e w d r o p s of toluene a d d e d , a n d the s o l u t i o n is stored i n the refrigerator at about 5 ° C . W e d o not find e v i d e n c e for surface d e n a t u r a t i o n of the h e m o c y a n i n s u n d e r the c o n d i t i o n s d e s c r i b e d for the p a r t i c u l a r h e m o c y a n i n e m p l o y e d .

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2

Since w e are w o r k i n g w i t h a b i o l o g i c a l fluid subject to d e t e r i o r a t i o n , i t is e s p e c i a l l y i m p o r t a n t that r e l i a b l e c r i t e r i a be e m p l o y e d to ensure that the o b s e r v e d responses of h e m o c y a n i n to c h e m i c a l a n d p h y s i c a l stress are r e a l a n d r e p r o d u c i b l e a n d not artefacts. T h e literature contains m a n y references to the storage life of h e m o c y a n i n . S o m e investigators h a v e c l a i m e d that the h e m o l y m p h c a n be stored for w e e k s or e v e n m o n t h s w i t h o u t d e t e r i o r a t i o n as l o n g as the m a t e r i a l is k e p t c o l d a n d c o v e r e d w i t h toluene. H o w e v e r , the q u e s t i o n arises as to the c r i t e r i a of d e t e r i o r a ­ t i o n e m p l o y e d . I n terms of i m m u n o l o g i c a l reactions (20), catalase activ­ i t y , a n d t o t a l o x y g e n c a p a c i t y , h e m o c y a n i n appears r e m a r k a b l y stable. W e h a v e f o u n d , h o w e v e r , b y o u r c r i t e r i a , that the refrigerator l i f e of o u r h e m o c y a n i n is a p p r o x i m a t e l y t w e l v e days. T h e c r i t e r i a w e e m p l o y is a t i m e d e p e n d e n c y of t h e o x y g e n a t e d a n d d e o x y g e n a t e d o p t i c a l density at 340 m/x. S o m e of the observations w e h a v e m a d e r e g a r d i n g the usa­ b i l i t y of h e m o c y a n i n i n h e m o l y m p h are: ( 1 ) T h e o x y g e n a t e d o p t i c a l density, ( O D ) , f o r a g i v e n d i l u t i o n , m a y b e a t t a i n e d e a c h d a y , b u t i n a d e t e r i o r a t e d sample, there occurs a spontaneous loss of o x y g e n u p o n s t a n d i n g 3 0 - 6 0 m i n u t e s w i t h a c o n ­ c o m i t a n t d r o p i n ( O D ) . U p o n r e o x y g e n a t i o n the ( O D ) m a y rise a g a i n but only temporarily. 0

0

0

( 2 ) T h e d e o x y g e n a t e d o p t i c a l density, ( O D ) , of a d e t e r i o r a t e d s a m p l e is h i g h e r t h a n that of a fresh p r e p a r a t i o n a n d m a y b e 2 5 - 5 0 % h i g h e r i n ( O D ) t h a n the f r e s h sample. d

d

( 3 ) T h e p H of f r e s h h e m o c y a n i n , Busycotypus h e m o c y a n i n i n its h e m o l y m p h or s e r u m is 8.1 ± 0.15 a n d that of Limulus is 7.6 ± 0.5. T h e p H undergoes s m a l l changes f r o m d a y to d a y , b u t the p H change i n a d e t e r i o r a t e d s a m p l e undergoes a l a r g e r t h a n n o r m a l d r o p . A n o r m a l decrease m a y b e f r o m 0.00 to 0.15 of a p H u n i t p e r d a y , b u t w h e n the samples deteriorate the p H d r o p s 0.3-0.5 of a u n i t . R e g u l a t i o n of h e l i u m gas f l o w t h r o u g h the v a r i o u s solutions are satisfactorily c o n t r o l l e d b y constant d i f f e r e n t i a l l o w flow controllers m a n u f a c t u r e d b y M o o r e P r o d u c t s C o . , P h i l a d e l p h i a , P a . B y this means w e are able to o b t a i n a constant flow of gas for d e o x y g e n a t i o n regardless of changes i n d o w n s t r e a m pressure. W e e m p l o y a u n i t c o n s i s t i n g of six

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

34.

SCHUBERT E T A L .

485

Hemocyanin

d i f f e r e n t i a l flow controllers c o n n e c t e d i n p a r a l l e l . E a c h u n i t c a n b e a d j u s t e d w i t h o u t a n y effect o n the others. W e find that 40 m l . of h e l i u m p e r m i n u t e p r o v i d e s a satisfactory flow f o r r a p i d d e o x y g e n a t i o n of d i l u t e solutions of o x y g e n a t e d h e m o c y a n i n . T h e flow rate is m e a s u r e d b y a s i m p l e soap b u b b l e meter. T h e r e o x y g e n a t i o n of d e o x y h e m o c y a n i n b y m o l e c u l a r o x y g e n is c o m p l e t e i n a m a t t e r of seconds. H o w e v e r , the d e o x y g e n a t i o n c y c l e i n o u r system r e q u i r e s m o r e t i m e . W i t h h e m o c y a n i n solutions d i l u t e d w i t h p h o s p h a t e buffer to a n o p t i c a l d e n s i t y of a b o u t 0.5, a n d a t o t a l v o l u m e of a b o u t 4 m l , d e o x y g e n a t i o n is c o m p l e t e i n a b o u t three m i n u t e s b u t m o r e c o n c e n t r a t e d solutions r e q u i r e m o r e t i m e . W e r o u t i n e l y d e o x y genate f o r six m i n u t e s w i t h h e m o c y a n i n s o l u t i o n of ( O D ) = 0.5 a n d ten m i n u t e s f o r solutions h a v i n g a n ( O D ) = 1. T h e rates of d e o x y ­ g e n a t i o n are l i t t l e affected b y the age, p H 5 - 9 , or degree of d i l u t i o n . 0

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch034

c

D i l u t i o n s are m a d e w i t h 0 . 0 5 M s o d i u m d i h y d r o g e n p h o s p h a t e b u f f e r , p H 7.00. C o p p e r analyses are m a d e b y a t o m i c a b s o r p t i o n s p e c t r o p h o ­ t o m e t r y ; the r a d i a t i o n source, cobalt-60 g a m m a rays, d e l i v e r s a b o u t 15,000 r a d / m i n . I n some cases 280 k v x-rays w e r e u s e d f o r i r r a d i a t i o n . R a d i a t i o n doses are m e a s u r e d w i t h a ferrous sulfate dosimeter b a s e d o n a v a l u e of G ( F e ) = 15.5 (28). F o r r a d i a t i o n doses a p p r o a c h i n g 40,000 rads w e use a n o x y g e n - s a t u r a t e d ferrous sulfate s o l u t i o n c o n t a i n i n g 4 m i l l i m o l e s of i r o n i n s t e a d of the u s u a l 1 m i l l i m o l e . I n this m a n n e r w e c a n e x t e n d t h e u s e f u l range to at least 10 rads. F o r r a d i a t i o n doses b e l o w 4,000 rads w e e m p l o y a n o p t i c a l c e l l w i t h a 10 c m . p a t h l e n g t h i n s t e a d of a 1 c m . p a t h l e n g t h . T h i s p e r m i t s us to o b t a i n r e l i a b l e measurements d o w n to 400 rads t o t a l a b s o r b e d dose. 3 +

5

O x y g e n - s a t u r a t e d solutions of h e m o c y a n i n are i r r a d i a t e d at 25 ° C . i n 4 m l . glass v i a l s sealed w i t h T e f l o n - l i n e d s c r e w caps. A f t e r i r r a d i a t i o n , o x y g e n a t e d solutions are t r a n s f e r r e d to s i l i c a c e l l cuvettes ( p a t h l e n g t h 1 c m . ) c a p p e d w i t h sleeve-type r u b b e r stoppers w i t h a n i n d e n t e d area i n the center so that s y r i n g e needles f o r passage of o x y g e n or oxygen-free h e l i u m c a n b e i n s e r t e d . H e m o c y a n i n is s u b s e q u e n t l y d e o x y g e n a t e d b y b u b b l i n g f r o m 350 to 600 m l . of h e l i u m , at a rate of 40 m l . p e r m i n u t e , t h r o u g h the s o l u t i o n u n t i l c o m p l e t e d e o x y g e n a t i o n occurs as m e a s u r e d b y the d e o x y g e n a t e d o p t i c a l d e n s i t y . T h e s o l u t i o n is k e p t i n the d e o x y ­ g e n a t e d state f o r a g i v e n t i m e , t h e n r e o x y g e n a t e d , a n d the o x y g e n a t e d o p t i c a l d e n s i t y is m e a s u r e d . S u b s e q u e n t measurements are m a d e u n t i l the ( O D ) reaches a constant v a l u e . T h e ( O D ) of h e m o c y a n i n f r o m Limulus attains a constant v a l u e i m m e d i a t e l y after o x y g e n a t i o n . W i t h i r r a d i a t e d h e m o c y a n i n f r o m Busycotypus, the t i m e r e q u i r e d f o r the ( O D ) to r e a c h a stable m a x i m u m v a l u e increases w i t h i n c r e a s i n g r a d i a t i o n dose —e.g., a dose of less t h a n 8,000 rads r e q u i r e s less t h a n one m i n u t e ; a p p r o x i m a t e l y 13,000 to 20,000, one h o u r ; 20,000 to 27,000, one to t w o h o u r s ; a n d three to f o u r h o u r s f o r doses a b o v e 34,000 rads. 0

0

G

A f t e r i r r a d i a t i o n , or sometimes before, s m a l l amounts of aggregated p r o t e i n m a y a p p e a r i n suspension i n the h e m o c y a n i n solutions a n d are r e m o v e d b y l o w s p e e d c e n t r i f u g a t i o n . T h e a g g r e g a t e d p r o t e i n is colorless a n d p r o b a b l y not h e m o c y a n i n because its r e m o v a l does not c h a n g e the o r i g i n a l O D ' s . H o w e v e r , t h e i r presence does raise the O D s l i g h t l y (.—0.05 a n O D u n i t ) . If the s u s p e n d e d aggregates a p p e a r t h e i r effect c a n b e

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

486

RADIATION CHEMISTRY

1

c o m p e n s a t e d for i n Limulus h e m o c y a n i n as f o l l o w s : d u r i n g d e o x y g e n a ­ t i o n the ( O D ) is r e c o r d e d . T h e s a m p l e is o x y g e n a t e d a n d the ( O D ) r e c o r d e d . T h e o x y g e n a t e d s a m p l e is e e n t r i f u g e d , a n d the ( O D ) a g a i n is r e a d . T h e difference i n ( O D ) b e t w e e n the e e n t r i f u g e d a n d n o n c e n t r i f u g e d sample is s u b t r a c t e d f r o m the d e o x y g e n a t e d sample O D to g i v e a c o r r e c t e d ( O D ) . T h e same p r o c e d u r e is u s e d w i t h Busycotypus h e m o c y a n i n for ( O D ) values, b u t because of r a p i d spontaneous reoxy­ g e n a t i o n f r o m r e s i d u a l H 0 , a c o r r e c t i o n to the ( O D ) is not feasible. d

0

0

G

d

0

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2

2

d

A n i m p o r t a n t factor, not a l w a y s a p p r e c i a t e d i n investigations of the effects of i o n i z i n g r a d i a t i o n o n b i o l o g i c a l materials i n aqueous s o l u t i o n , is the d e p l e t i o n of o x y g e n i n the m e d i u m w h i c h occurs d u r i n g i r r a d i a t i o n . I n o u r systems, this factor becomes i m p o r t a n t e v e n i n o x y g e n saturated s o l u t i o n above doses of 60,000 rads a n d at l o w e r doses w i t h air saturated solutions. F r o m experience, w e f o u n d that it is necessary either to b u b b l e o x y g e n t h r o u g h the s o l u t i o n d u r i n g i r r a d i a t i o n or to m a i n t a i n the o x y g e n saturated s o l u t i o n i n a closed system d u r i n g i r r a d i a t i o n . I n order to check w h e t h e r o u r system has suffered o x y g e n d e p l e t i o n at h i g h r a d i a t i o n doses, w e measure the ( O D ) of a n i r r a d i a t e d o x y g e n a t e d h e m o c y a n i n s o l u t i o n i m m e d i a t e l y after i r r a d i a t i o n a n d b e f o r e f u r t h e r o x y g e n a t i o n . If i t de­ creases t h e n w e k n o w that d e p l e t i o n o c c u r r e d . I n a s m u c h as o n l y the f u l l y o x y g e n a t e d h e m o c y a n i n is resistant to the a c t i o n of the r a d i a t i o n p r o d u c e d p r o d u c t s , it is o b v i o u s l y necessary that no o x y g e n d e p l e t i o n o c c u r i n the system d u r i n g i r r a d i a t i o n . G

H y d r o g e n p e r o x i d e is f o r m e d i n the i r r a d i a t e d o x y g e n a t e d buffer solutions a n d the concentrations w e r e m e a s u r e d i o d o m e t r i c a l l y ( 8 ) i n p H 7 p h o s p h a t e buffer. A m o u n t s of H 0 f o r m e d w e r e , i n /xmole/liter: 18 at 10 k r a d , 26 at 15 k r a d , 58 at 40 k r a d s . I n d e o x y g e n a t e d buffer n o H 0 was detected ( < 1 /xmole/liter ). T h e results are i n g o o d agree­ m e n t w i t h other w o r k e r s ( 5 ). 2

2

2

2

T h e c o p p e r i n h e m o c y a n i n is d i r e c t l y a n d q u a n t i t a t i v e l y i n v o l v e d i n the o x y g e n a t i o n r e a c t i o n . A l l i n c o m i n g h e m o c y a n i n solutions are a n a l y z e d f o r c o p p e r w i t h a P e r k i n - E l m e r a t o m i c a b s o r p t i o n spectrophotometer. T h e analyses are c a r r i e d out b y d i l u t i n g the stock s o l u t i o n w i t h d i s t i l l e d w a t e r a n d f e e d i n g t h e s o l u t i o n to the b u r n e r . T h e catalase-like properties of h e m o c y a n i n a n d c o p p e r - a m i n o a c i d chelates h a v e b e e n m e a s u r e d m a n o m e t r i c a l l y u s i n g a c o n v e n t i o n a l W a r ­ b u r g a p p a r a t u s . W e are n o w u s i n g a d i f f e r e n t i a l m a n o m e t e r t e c h n i q u e f o r m e a s u r i n g the rates of o x y g e n e v o l u t i o n (31 ) w h i c h , f o r o u r purposes, is m u c h s u p e r i o r to the c o n v e n t i o n a l v e r t i c a l c o l u m n d i f f e r e n t i a l m a n o m e ­ ter, e s p e c i a l l y i n sensitivity. Results N o effect of i r r a d i a t i o n o n h e m o c y a n i n is d e t e c t e d as l o n g as the h e m o c y a n i n is m a i n t a i n e d i n the o x y g e n a t e d state. H o w e v e r , u p o n d e o x y g e n a t i o n of the i r r a d i a t e d h e m o c y a n i n , f o l l o w e d b y o x y g e n a t i o n , a decrease i n o x y g e n - c a r r y i n g c a p a c i t y is o b s e r v e d . T h e a m o u n t of r a d i a ­ t i o n d a m a g e — m e a s u r e d i n terms of loss of o x y g e n c a p a c i t y — d e p e n d s o n the t i m e the i r r a d i a t e d h e m o c y a n i n r e m a i n s i n the o x y g e n a t e d state

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

34.

SCHUBERT E T A L .

487

Hemocyanin

( d e s i g n a t e d as o x y g e n r e a c t i o n t i m e , o.r.t.) b e f o r e a d e o x y g e n a t i o n a n d r e o x y g e n a t i o n c y c l e , a n d i n the d e o x y g e n a t e d state ( d e s i g n a t e d as deoxy­ genated r e a c t i o n t i m e , d.r.t. ) b e f o r e r e o x y g e n a t i o n .

H o w e v e r , u n d e r the

c o n d i t i o n s e m p l o y e d , the r a d i a t i o n effects are i n d e p e n d e n t of the o.r.t.'s f o r the t i m e intervals e m p l o y e d . a n d the d.r.t. w a s one h o u r . w i t h an ( O D )

0

G e n e r a l l y , the o.r.t. w a s t e n m i n u t e s

I n the case of Limulus

hemocyanin—e.g.,

of 0.5—the effect of i r r a d i a t i o n o n the ( O D )

0

was identi­

c a l f o r o.r.t.'s u p to 60 m i n u t e s after the cessation of i r r a d i a t i o n . Samples of Limulus

a n d Busycotypus

were diluted w i t h

phosphate

buffer to y i e l d d e s i r e d values of o x y g e n a t e d o p t i c a l density. T h e h e m o ­ Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch034

c y a n i n w a s i r r a d i a t e d i n the o x y g e n a t e d state, d e o x y g e n a t e d i m m e d i a t e l y after i r r a d i a t i o n , t h e n reoxygenated.

The ( O D )

G

decreased w i t h increas­

i n g dose a n d the G ( - 0 ) values ( 0 . 9 - 1 . 2 ) , f o r b o t h h e m o c y a n i n s 2

were

about the same a n d i n d e p e n d e n t of c o n c e n t r a t i o n ( F i g u r e 1 ). I n F i g u r e 2 d a t a for a one h o u r d.r.t. ( a p e r i o d b e y o n d w h i c h n o f u r t h e r decreases i n o x y g e n c a p a c i t y o c c u r ) s h o w that the ( O D )

G

drops

r a p i d l y w i t h i n c r e a s i n g r a d i a t i o n a n d , i n fact, m o r e t h a n 9 0 % of the entire o x y g e n c a p a c i t y is lost above 35,000 rads. E x p e r i m e n t s w i t h Busycotypus to those o b t a i n e d w i t h Limulus

h e m o c y a n i n y i e l d e d results s i m i l a r

hemocyanin.

However, w i t h an

(OD)

0

of 0.5, r a d i a t i o n doses a b o v e 13,000 to 16,000 rads restored the o x y g e n capacity,

and above

70,000 rads the o x y g e n c a p a c i t y

e q u i v a l e n t to u n i r r a d i a t e d h e m o c y a n i n ( 3 7 ) . of h e m o c y a n i n w a s i n c r e a s e d to give a n ( O D )

0

23,000 rads, c a l c u l a t e d = M X 13,000 = 0.5

nearly

concentration

of 0.9, the r a d i a t i o n dose

r e q u i r e d to p r o d u c e the m i n i m u m i n c r e a s e d to that =

became

W h e n the

expected—observed

23,000 rads ( F i g u r e 3 ) .

Role of Radiolytic Hydrogen Peroxide. A series of experiments w e r e c a r r i e d out to d e t e r m i n e the extent to w h i c h r a d i o l y t i c H 0 2

2

contributed

to the decrease i n o x y g e n c a p a c i t y of i r r a d i a t e d h e m o c y a n i n . F r o m these experiments s u m m a r i z e d b e l o w , the c o n c l u s i o n is d r a w n that the effects of g a m m a i r r a d i a t i o n of h e m o c y a n i n i n solutions of h e m o l y m p h or buffer i n the dose range of 0 to 60 k r a d s are c a u s e d n e a r l y e n t i r e l y b y

H 0 . 2

2

T h e e x p e r i m e n t a l bases for this c o n c l u s i o n f o l l o w : ( 1 ) T h e p h o s p h a t e buffer was i r r a d i a t e d a n d a d d e d to u n i r r a d i a t e d h e m o c y a n i n . T h e subsequent changes i n o x y g e n - c a r r y i n g c a p a c i t y w e r e i d e n t i c a l w i t h that o b s e r v e d w h e n the h e m o c y a n i n w a s i r r a d i a t e d w h i l e i n the same buffer over the entire range of r a d i a t i o n doses. B o t h Limulus a n d Busycotypus h e m o c y a n i n w e r e tested. I n the case of the latter, e v e n the regeneration of o x y g e n - c a r r y i n g c a p a c i t y w a s r e p r o d u c e d ( F i g u r e 4 ) . ( 2 ) T h e presence of catalase d u r i n g i r r a d i a t i o n of h e m o c y a n i n o r the a d d i t i o n of catalase to h e m o c y a n i n after i r r a d i a t i o n , b u t b e f o r e d e ­ o x y g e n a t i o n , eliminates the effects of i r r a d i a t i o n o n the o x y g e n - c a r r y i n g capacity (Figure 5).

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

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488

RADIATION CHEMISTRY

4

6

1

8

RADIATION DOSE (KIL0RADS)

Figure 1. Effect of gamma irradiation on the oxygencarrying capacity of different concentrations of oxy­ genated hemocyanins in phosphate buffer at pH 7.0. The oxygenation reaction time was 10 minutes and the deoxygenation reaction time was 30 minutes Busycotypus 0 — [ C w ] = 7.1 X I 0 - M ; G ( - O * ) r = 1 . 2 5

• — [ C M ] = 5 . 3 5 Χ 10~ Μ; G ( - C \ ) = 5

1.0

O — [ C M ] = 3.55 X 10- M; G(-0 ) = 0.9 Limulus • — [ C M ] = 7.68 X 10- M; G(-0,) = 1.1 # — [ C M ] = 4.04 X 10~ M; G(-O ) = 0.9 5

2

5

5

t

( 3 ) T h e h e m o l y m p h was separated f r o m hemocyanin b y ultracent r i f u g a t i o n . W h e n t h e protein-free h e m o l y m p h w a s i r r a d i a t e d a n d a d d e d to u n i r r a d i a t e d b u f f e r e d ( p h o s p h a t e ) h e m o l y m p h , the effect w a s i d e n t i c a l w i t h t h a t o b s e r v e d f o r the same doses of i r r a d i a t i o n d e l i v e r e d t o h e m o ­ c y a n i n i n h e m o l y m p h . T h e a m o u n t of H 0 p r o d u c e d i n h e m o l y m p h w a s f o u n d to b e t h e same as that p r o d u c e d i n p u r e buffer f o r t h e r a d i a t i o n dose range, 0 - 6 0 k r a d s . 2

2

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

34.

SCHUBERT E T A L .

489

Hemocyanin

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch034

0.5

20

30

40

ABSORBED

50

60

RADIATION

DOSE

70

80

90

100

(KILORADS) Science

Figure 2. Changes in optical densities of hemocyanin from L i m u l u s after irradiation at 25°C. with cobalt-60 gamma rays at a dose rate of 15 kilorads/ min. Solutions consisted of L i m u l u s hemolymph diluted with 0.05M potassium dihydrogen phosphate buffer (pH 7.00) and saturated with oxygen. Copper concentration was 3.92 X 10~ M. Hemocyanin was deoxygenated ten minutes after irradiation and was allowed to remain in the deoxygenated state for one hour before reoxygenation, at which time the optical densities were measured at 340 ra/x. Subsequent deoxygenation provided the final deoxygenated optical densities. 5

Ο—oxygenated φ—deoxygenated

( 4 ) T h e a d d i t i o n of H 0 to b u f f e r e d solutions of h e m o c y a n i n p r o ­ d u c e d effects v e r y s i m i l a r to those o b s e r v e d u p o n i r r a d i a t i o n . T h e s e results a l o n g w i t h those of i r r a d i a t e d systems, also reflect s m a l l effects c a u s e d b y t h e c a t a l y t i c d e c o m p o s i t i o n of H 0 b y h e m o c y a n i n itself. 2

2

2

Effect of

O r g a n i c Peroxides.

properties of Limulus tested.

T h e effect o n t h e o x y g e n - c a r r y i n g

h e m o c y a n i n b y different o r g a n i c peroxides w a s

T h e peroxides tested i n c l u d e d : s u c c i n i c a c i d p e r o x i d e

C H — C H — C O ) 0 ; tert-butyl 2

2

2

2

2

hydroperoxide ( ( C H

3

(HOOC—

) « C — O O H ); and

Ο tert-butyl

peroxymaleie acid ( ( C H

A t t h e highest concentrations

H

) C — O O — C — C H = C H — C O O H ). 3

tested w h i c h i n c l u d e d levels h i g h e r t h a n

that e m p l o y e d w i t h H 0 , n o effects o n o x y g e n - c a r r y i n g w e r e o b s e r v e d . It 2

2

appears that the size a n d shape of the h y d r o c a r b o n side chains a r o u n d the p e r o x i d e o x y g e n are a c r i t i c a l factor since t h e y d e t e r m i n e t h e a b i l i t y

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

490

RADIATION CHEMISTRY

to contact p r o t e i n - b o u n d c o p p e r . b e i n g e x t e n d e d to Busycotypus Effect of

T h e s e studies are c o n t i n u i n g a n d are

hemocyanin. pH.

Urea, Calcium, and

c h e m i c a l or r a d i o l y t i c H 0 2

2

When

the

concentration

exceeds that of the c o p p e r i n

h e m o c y a n i n , the o x y g e n - c a r r y i n g c a p a c i t y is regenerated. true of Limulus

1

of

Busycotypus T h i s is not

h e m o c y a n i n , p r e s u m a b l y c a u s e d b y the relative inaccessi­

b i l i t y of the p r o t e i n - b o u n d c o p p e r . u p " the structure of Limulus

A c c o r d i n g l y w e a t t e m p t e d to " o p e n

b y the use of h i g h ( 6-7 M ) solutions of u r e a

w h i c h c o u l d cause the p r o t e i n structure to u n f o l d . H o w e v e r , urea, u n d e r the c o n d i t i o n s tested, h a d l i t t l e or no effect o n either the o x y g e n - c a r r y i n g c a p a c i t y or H 0

2

regeneration of Limulus

hemocyanin.

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2

10

20

30

40

50

RADIATION

60

70

80

90

DOSE (KILORADS)

Figure 3. Effect of gamma radiation on oxygenated Busycotypus hemo­ cyanin in phosphate buffer, pH 7 at 25°C. Copper concentration was 7.1 X 10~ M 5

Ο—(OD)o at • —(OD)o at •—(OD)d at #—(OD) at d

1 hr. deoxygenated reaction time (d.r.t.) 15 min. d.r.t. 15 min. d.r.t. 1 hr. d.r.t.

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

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34.

SCHUBERT E T A L .

0

10

20

30

40

RADIATION

Figure 4.

491

Hemocyanin

50

DOSE

60

70

80

90

(KILORADS)

Medium effects on hemocyanin. Effect of oxygenated gamma irradi­ ated, 0.05M, buffer (pH 7) on unirradiated hemocyanin

0 , Ο Hemocyanin and buffer irradiated together Q , Δ Irradiated buffer added to unirradiated hemocyanin N e i t h e r the o x y g e n a t i o n reactions of Limulus d e s t r u c t i o n of o x y g e n - c a r r y i n g c a p a c i t y b y H 0 2

2

h e m o c y a n i n n o r the w e r e affected b y v a r y ­

i n g p H ( 5 . 5 - 8 ) a n d a d d e d c a l c i u m levels ( 0 . 0 0 5 6 - 0 . 0 3 2 M ).

Whether

these p a r t i c u l a r experiments a c t u a l l y p r o d u c e d changes i n the state of a g g r e g a t i o n of the h e m o c y a n i n w a s not tested. C a t a l y t i c D e c o m p o s i t i o n of H 0 . 2

decomposes H 0 2

2

2

Hemocyanin from

a n d releases o x y g e n (10).

Busycotypus

W e m e a s u r e d this c a t a l y t i c

a c t i o n o n samples of h e m o c y a n i n h e a t e d for 15 m i n u t e s at different t e m ­ peratures a n d p H 7.

T h e g e n e r a l effectiveness

of the c a t a l y t i c a c t i o n

w a s r e l a t i v e l y u n c h a n g e d u p to temperatures as h i g h as 6 5 ° C . ( F i g u r e 6 ) . T h e c a t a l y t i c effectiveness of Limulus f r o m Busycotypus pH

(Figure 6).

h e m o c y a n i n is far l o w e r t h a n that

a n d was m a n i f e s t e d o n l y at h i g h e r concentrations a n d I r r a d i a t i o n of h e m o c y a n i n w i t h doses u p to 100 k r a d s

d i d not affect the c a t a l y t i c a c t i v i t y . T h e catalase-like a c t i o n of h e m o c y a n i n is p r o b a b l y because of c o p p e r b o u n d to one or m o r e a m i n o acids i n the p r o t e i n . C o n t r a r y to p r e v i o u s c l a i m s (12),

a r g i n i n e chelates w i t h c o p p e r are not the o n l y c a t a l y t i c a l l y

active species. F o r e x a m p l e , c o p p e r chelates w i t h h i s t i d i n e a n d h i s t a m i n e are also active. T h e rates a p p e a r to be a first p o w e r f u n c t i o n of c o p p e r and H 0 . 2

2

Studies n o w b e i n g c a r r i e d out w i t h V . S. S h a r m a i n o u r

laboratories i n d i c a t e that the a c t i v e species is the C u ( I I ) L f o r m w h e r e L represents the l i g a n d . T h e c o p p e r chelate f o r m s a t e r n a r y c o m p l e x w i t h

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

492

RADIATION CHEMISTRY

the H O O " a n i o n of H 0 2

w h i c h s u b s e q u e n t l y decomposes to 0

2

radical and molecular mechanisms.

2

1

b y free

T h e most c a t a l y t i c a l l y active l i g a n d s

i n v o l v e the c o o r d i n a t i o n o f t w o n i t r o g e n atoms to c o p p e r . I t is a n t i c i p a t e d that t h e studies w i t h m o d e l c o p p e r complexes m a y c l a r i f y t h e m o d e of attachment o f c o p p e r to different h e m o c y a n i n s .

ζ < ο ο Σ

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Lu I

ο

0.5 I 0

I 10

I I I I 20 30 40 50 RADIATION DOSE (KILORADS)

I

I

60

70

Figure 5. Effect of catalase on response of Busycotypus hemocyanin to gamma irradiation. Catalase concentration was 20 μg/ml. •, Φ Catalase present during irradiation or added afterwards A No catalase added Discussion The

r e a c t i v a t i o n o f Busycotypus

n a t u r e of H 0 2

2

h e m o c y a n i n illustrates t h e d u a l

n a m e l y , that i t is also a r e d u c i n g agent (38, 44). T h e

r a d i a t i o n r e a c t i v a t i o n of Busycotypus

h e m o c y a n i n , at t h e concentrations

e m p l o y e d , is b r o u g h t a b o u t b y t h e r e d u c t i o n of C u ( I I ) to C u ( I ) b y H 0 2

2

as occurs w i t h H 0 2

2

a d d e d to u n i r r a d i a t e d h e m o c y a n i n

Some of t h e r e d o x reactions c o p p e r c a n b e d e d u c e d (3, 5,44)

(10).

that m a y i n v o l v e t h e p r o t e i n - b o u n d i f w e consider that w e are d e a l i n g w i t h

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

34.

SCHUBERT E T A L .

τ

493

Hemocyanin

1

1

1

1

1

Γ

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ο

0

ΙΟ

20

30

40

TIME

50

60

70

80

(MINUTES)

Figure 6, Liberation of oxygen from hydrogen peroxide in the presence of Busycotypus hemocyanin at pH 7 but at different temperatures and of L i m u l u s hemocyanin at 25°C. and pH 9.5. The Busycotypus hemocyanin solutions were heated for 15 minutes at a given temperature prior to mixing with hydrogen peroxide Busycotypus hemocyanin Warburg flask contained 14.4 ^moles H O (0.2 ml. of 0.072M), 0.5 ml. of hemo­ cyanin serum (7.5 X I O M in copper) and 0.2 cc. of 0.05M pH 7 phosphate buffer. 1 mm. = 0.062 ^moles O : Solid line, 25°C; O , 35°C; C L 45°C.;, 65°C; 65°C. and deoxygenated while heated Limulus hemocyanin Conditions as for Busycotypus except that the copper concentration was 2.0 X 10~ M, pH 9.5, and temperature of 25°C. t

t

_i

t

3

a n e u t r a l , o x y g e n a t e d , aqueous m e d i u m c o n t a i n i n g o r g a n i c solutes a n d that r e d u c t i o n takes p l a c e m o r e r e a d i l y i n n e u t r a l t h a n i n a c i d m e d i a . F r o m R e f e r e n c e 37 t h e most l i k e l y reactions i n o x y g e n a t e d m e d i a i n c l u d e : (1) Oxidation ( inactivation) P-Cu(I) + H 0 2

2

= P-Cu(II) + O H + O H "

P-Cu(I) + O H = P-Cu(II) + O H "

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

494

RADIATION CHEMISTRY

1

(2) Reduction (reactivation) P-Cu(II) + H 0 = P-Cu(I) + H 0 + H 2

2

2

P-Cu(II) + H 0 = P-Cu(I) + 0 2

Whether

the radiation

2

doses employed modify the hemocyanin

molecule is a question which we are exploring. Limulus

+

hemocyanin in the deoxygenated

W e have

irradiated

state at fairly high doses

(^100,000 rads) and have found little change in the oxygenation-deoxygenation properties.

W e have also begun to use other techniques with

which to ascertain possible effects of ionizing radiation on the hemo­ Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch034

cyanin molecule including optical rotatory dispersion and circular d i chroism (46).

For the present, it appears that the principal effect of ioniz­

ing radiation on hemocyanin is centered on the active copper sites through the promotion of the oxidation of the C u ( I ) to the C u ( I I ) state by radiolytic hydrogen peroxide. Acknowledgment This investigation was supported by U . S. Public Health Service Research Grant R H 00434, National Center for Radiological Health and by A E C contract A T ( 30-1)-3641.

Literature Cited (1) Anbar, M., Nature 200, 376 (1963). (2) Bacq, A. M., Alexander, P., "Fundamentals of Radiobiology," Pergamon Press, New York, 1961. (3) Barb, W. G., Baxendale, J. H., George, P., Hargrave, K. R., Trans. Fara­ day Soc. 47, 462, 591 (1951). (4) Barron, E. S. G., Ann. New York Acad. Sci. 59, 574 (1955). (5) Baxendale, J. H., Radiation Res. Suppl. 4, 114 (1964). (6) Bayer, E., Liebigs Ann. Chem. 653, 149 (1962). (7) Cohen, L. B., Van Holde, Κ. E., Biochemistry 3, 1809 (1964). (8) Egerton, A. C., Everett, A. J., Minkoff, G. J., Rudrakanchana, S., Salooja, K. C., Anal. Chim. Acta 10, 422 (1954). (9) Felsenfeld, G.,J.Cellular Comp. Physiol. 43, 23 (1954). (10) Felsenfeld, G. Printz, M. P.,J.Am. Chem. Soc. 81, 6259 (1959). (11) Frieden, E., Osaki, S., Kobayashi, H., J. Gen. Physiol. 49, 213 (1965). (12) Ghiretti, F., Arch. Biochem. Biophys. 63, 165 (1956). (13) Ghiretti, F.,"Oxygenases,"Chap. 10, O. Hayaishi, ed., Academic Press, New York, 1962. (14) Ghiretti-Magaldi, A. Nuzzolo, Ghiretti, F., Biochemistry 5, 1943 (1966). (15) Hayaishi, O. (ed.), "Oxygenases," Academic Press, New York, 1962. (16) Hudson, T. A. F., "Vanadium-Toxicology and Biological Significance," Elsevier Publishing Co., New York, 1964. (17) Larimer, J. L., Riggs, A. F., Comp. Biochem. Physiol. 13, 35 (1964). (18) Johnston, W., James, T. W., and Barber, Α. A., Comp. Biochem. Physiol. 22, 261 (1967). (19) Levitzki, Α., Anbar, M., J. Am. Chem. Soc. 89, 4185 (1967). (20) Litt, M., Boyd, W.C.,Nature 181,1075(1958).

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

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34.

SCHUBERT

E T A L .

Hemocyanin

495

(21) Lontie, R. Blaton, V., Albert, M., Peeters, B., Arch. Intern. Physiol. Biochim. 73, 150 (1965). (22) Lontie, R., Witters, R., "The Biochemistry of Copper," pp. 455-462, J. Peisach, P. Aisen, W. E. Blumberg, eds., Academic Press, New York, 1966. (23) Manwell,C.,Ann. Rev. Physiol. 22, 191 (1960). (24) Mason, H. S., Nature 177, 79 (1956). (25) Mason, H. S., Advan. Enzymol. 19, 79 (1957). (26) Nagy-Keresztes, S., Klotz, I. M., Biochemistry 4, 919 (1965). (27) Nakamura, T., Mason, H. S., Biochem. Biophys. Res, Comm. 3, 297 (1960). (28) Natl. Bur. Std. (U. S.) Handbook 85, 14 (1964). (29) Nikolaev, L. Α., "The Origin of Life on the Earth," pp. 263-274, F. Clark, R. L. M. Synge, eds., Pergamon Press, New York, 1959. (30) Orgel, L. E., In Metals and Enzyme Activity, Symposium No. 15, Cam­ bridge, 1958. (31) Peterson, R. N., Freund, M., Gilmont, R., Proc. Soc. Exp. Biol. Med. 125, 645 (1967). (32) Pickels, E. G., Anderson, R. S.,J.Gen. Physiol. 30, 83 (1946). (33) Redfield, A. C., "Copper Metabolism," W. D. McElroy, B. Glass, eds., The Johns Hopkins Press, Baltimore, 1950. (34) Schubert, J., Nature195,1096(1962). (35) Ibid.,200,375(1963). (36) Schubert, J., "Copper and Peroxides in Radiobiology and Medicine," C. C. Thomas, Springfield, Ill., 1964. (37) Schubert, J., White, E. R., Science 155, 1000 (1967). (38) Schumb, W. C., Satterfield, C. N., Wentworth, R. L., "Hydrogen Per­ oxide," p. 355, Reinhold, New York, 1955. (39) Shchepot'yeva, E. S., Ardashnikov, S. N., Lur'ye, G. E., Rakhamanova, T. B., "Effect of Oxygen in Ionizing Radiation," State Publishing House for Medical Literature, Moscow, 1959 (Translation, U. S. At. Energy Comm., Technical Information Service, AEC-tr-4265). (40) Sigel, H., Muller, V., Helv. Chim. Acta 49, 671 (1966). (41) Svedberg, T., Brohult, S., Nature 142, 830 (1938). (42) Ibid.,143,938(1939). (43) Swallow, A. J., "Radiation Chemistry of Organic Compounds," Pergamon Press, New York, 1960. (44) Uri, N., Chem. Rev. 50, 375 (1952). (45) Van Holde, Κ. E., Cohen, L. B., Biochemistry 3, 1803 (1964). (46) Van Holde, Κ. E., Biochemistry 6, 93 (1967). RECEIVED February 12,

1968.

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.