Effect of Î³-Irradiation on Aqueous Solutions of the Cyclic

33 Effect of γ-Irradiation on Aqueous Solutions

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of the Cyclic Dodecapeptide Polymyxin B J. KOPOLDOVA Isotope Laboratory of the Institutes for Biological Research, Czechoslovak Academy of Sciences, Prague, Czechoslovakia

As a continuation of our work on γ-irradiation of simple di- and tripeptides we have studied the radiation behavior of a more complex model—the cyclic, antibiotically active dodecapeptide polymyxin B—by irradiating its 0.5% aque ous solutions in oxygenated and oxygen-free atmospheres at doses of 1.36 X 10 -131.7 X 10 rad. The radiation prod ucts were determined in the irradiated solutions and in their hydrolysates, as well as the decrease of the content and biological activity of polymyxin. Both of these amounted to 45% at a dose of 13.7 X 10 rad. It was found that the main radiation mechanisms in solutions of polymyxin corre spond, in general, to those described for the radiolysis of simple peptides. The major reactions in oxygenated solutions are oxidative deamination, Garrison's cleavage of peptide bonds, and oxidative reactions in the amino acid residues. In oxygen-free media, reductive deamination and recombina tion reactions are the main processes. 5

5

5

T n h i t h e r t o p u b l i s h e d w o r k o n the effect of y - i r r a d i a t i o n o n a c e t y l a t e d A

a m i n o acids ( 3 ) a n d s i m p l e d i - a n d t r i p e p t i d e s (5, 9 ) , the f o l l o w i n g

m a i n r a d i o l y s i s m e c h a n i s m s of these substances h a v e b e e n established. In oxygenated m e d i a : ( 1 ) O x i d a t i v e d e a m i n a t i o n reactions of free - N H groups w h i c h p r o c e e d via t h e f o r m a t i o n of i m i n o acids a n d t h e i r h y d r o l y s i s a n d l e a d to t h e f o r m a t i o n o f k e t o a c y l p e p t i d e s o r to the f o r m i n g of t h e C - t e r m i n a l a m i n o a c i d a c c o r d i n g to t h e s u m m a r y scheme 2

N H C H (R ) C O N H C H (R ) C O O H + H 0 2

x

NH

3

2

2

2

+ H 0 -> 2

+ O = C (Ri) C O N H C H ( R ) C O O H 2

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

(1)

33.

KOPOLDOVA

Cyclic

Dodecapeptide

Polymyxin

NH CH(R )CONHCH(R )COOH + H 0 1

2

2

473

B

2

2

+ 2 H 0 -> 2

NH + C 0 + R i C H O + N H C H ( R ) C O O H 2

3

2

(2)

2

( 2 ) S i m u l t a n e o u s l y , o x i d a t i o n reactions take p l a c e o n t h e m e t h y l a n d m e t h y l e n e groups of the a m i n o a c i d chains w i t h the f o r m a t i o n of the c o r r e s p o n d i n g - O H , - C O , a n d - C O O H derivatives. I n peptides c o n t a i n i n g a m i n o acids w i t h b r a n c h e d chains or a r o m a t i c or h e t e r o c y c l i c n u c l e i , s p l i t t i n g of the c a r b o n c h a i n of these a m i n o a c i d residues takes place.


( 3 ) C l e a v a g e of the p e p t i d e b o n d a c c o r d i n g to G a r r i s o n ' s r e a c t i o n , w h i c h c a n b e s u m m a r i l y represented b y t h e scheme RCONHCHR

2

RCONHCR

+ 0

2

+ OH -» RCONHCR 2

2

+ H 0 -» RCONH 2

+ H 0

(3)

2

+ RCOR + H 0

2

2

(4)

I n oxygen-free m e d i a these reactions are suppressed a n d t h e processes taking place i n such m e d i a are: ( a ) R e d u c t i v e d e a m i n a t i o n of free - N H groups l e a d i n g to a m m o n i a a n d t h e f o r m a t i o n of a c y l peptides a c c o r d i n g to t h e scheme 2

H + H NCH(R )CONHCH(R )COOH-^ 1

2

2

N H + CH (Rj) C O N H C H ( R ) C O O H

(5)

2

3

CH ( R ) C O N H C H ( R ) C O O H + N H C H ( R ) C O N H C H ( R ) COOH -> x

2

2

x

2

C H (R )C O N H C H (R ) C O O H + N H C (R ) C O N H C H (R ) C O O H 2

x

2

2

x

2

(6)

( b ) R e c o m b i n a t i o n reactions w i t h t h e f o r m a t i o n of n e w — C — C — b o n d s , w h i c h y i e l d peptides of h i g h e r m o l e c u l a r w e i g h t . ( c ) T h e cleavage of p e p t i d e b o n d s , w h i c h i n these m e d i a takes p l a c e a c c o r d i n g t o t h e scheme H + R C O N H C H ( R ) C O O H -> R C O N H , + C H ( R ) C O O H -> R C O + N H C H ( R ) C O O H 2

(7) (8)

T h e p u r p o s e of t h e present w o r k w a s to investigate w h e t h e r the m e c h a n i s m s o u t l i n e d above also take p l a c e w h e n m o r e c o m p l e x p e p t i d e s c o n t a i n i n g a larger n u m b e r of p e p t i d e b o n d s are i r r a d i a t e d . A p e p t i d e w h i c h contains 10 to 12 a m i n o acids a n d is a stable, c h e m i c a l l y d e f i n e d substance, soluble i n water, a p p e a r e d to b e best to use f o r this p u r p o s e . F o r these reasons, chosen.

the peptide antibiotic p o l y m y x i n B

(sulfate) was

It possesses t h e f u r t h e r advantage that i n a d d i t i o n to c h e m i c a l

changes caused b y r a d i a t i o n , i t a l l o w s changes to f o l l o w i n its b i o l o g i c a l activity. P o l y m y x i n B ( C H 9 8 0 i N i m . w . 1 2 0 2 ) is a c y c l i c p e p t i d e c o n s i s t i n g 56

f o r the major part of L - ,

3

6

y - , d i a m i n o b u t y r i c a c i d ( 5 r e s i d u e s ) , aside of

L-threonine ( 2 ) , L-leucine ( 1 ) , D-phenylalanine ( 1 ) , and (-f)-6-methyloctanoic a c i d ( 1 ) . I n order to b e able to e x p l a i n t h e f o r m a t i o n of c e r t a i n d e g r a d a t i o n p r o d u c t s of this p e p t i d e i t w a s first necessary to o b t a i n some

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

474

RADIATION CHEMISTRY

1

b a s i c i n f o r m a t i o n o n the r a d i a t i o n c h e m i c a l b e h a v i o r of free d i a m i n o b u t y r i c a c i d w h o s e r a d i o l y s i s has n o t y e t b e e n d e s c r i b e d .


Experimental

Procedure

0 . 0 5 M aqueous solutions of free d i a m i n o b u t y r i c a c i d ( p r e p a r e d b y I n g . P o d u s k a , Institute of O r g a n i c C h e m i s t r y a n d B i o c h e m i s t r y , C z e c h o slovak A c a d e m y of S c i e n c e s ) a n d 0 . 5 % aqueous solutions of p o l y m y x i n B (sulfate) (Burroughs, W e l l c o m e and C o . ) were prepared. T h e s e solutions w e r e i n glass a m p u l e s i r r a d i a t e d either i n oxygenfree ( t h e a m p u l e s h a v i n g b e e n b u b b l e d w i t h n i t r o g e n a n d sealed p r i o r to i r r a d i a t i o n ) o r i n o x y g e n a t e d atmospheres ( i n the latter case o x y g e n w a s b u b b l e d t h r o u g h the a m p u l e s d u r i n g i r r a d i a t i o n ) . T h e r a d i a t i o n source w a s a C o d e v i c e of 2.7 X 1 0 r a d . / h r . i n t e n sity. F o r p o l y m y x i n B solutions t h e doses w e r e 1.36 X 1 0 , 13.17 X 1 0 , 66 X 1 0 , a n d 131.7 X 1 0 r a d . T h e solutions of free d i a m i n o b u t y r i c a c i d w e r e i r r a d i a t e d at a single dose of 131.7 X 1 0 r a d . , w h i c h w a s u s e d i n earlier w o r k f o r i r r a d i a t i n g free a m i n o acids a n d peptides. T h e decrease of t h e b i o l o g i c a l a c t i v i t y of i r r a d i a t e d p o l y m y x i n B solutions w a s d e t e r m i n e d b y t h e d i f f u s i o n a l m i c r o b i o l o g i c a l m e t h o d . A s a test c u l t u r e , w e u s e d Bacillus subtilis A T C C 633 a n d Eschericchia coli N C I B 8113. T h e experiments w e r e q u a n t i t a t i v e l y e v a l u a t e d b y the m e t h o d of R i e i e o v a a n d P o d o j i l (11). T h e c u l t u r e w a s c u l t i v a t e d a c c o r d i n g to the m e t h o d of Goss a n d K a t z (4). I n a l l the i r r a d i a t e d samples, free a n d a m i d e - l i k e a m m o n i a w e r e d e t e r m i n e d b y a m o d i f i c a t i o n of C o n w a y ' s m e t h o d (10) a n d c a r b o n y l substances w e r e d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y (8). T h e d e c o m p o s i t i o n p r o d u c t s i n i r r a d i a t e d solutions of p o l y m y x i n a n d i n their hydrolysates w e r e i d e n t i f i e d b y p a p e r electrophoresis i n a p y r i d i n e - a c e t a t e buffer s o l u t i o n ( p H 5.7, 1200 v o l t s ) a n d b y p a p e r c h r o m a t o g r a p h y u s i n g t h e systems b u t y l alcohol-acetic a c i d - w a t e r ( 4 : 1 : 5 ) and phenol-ethyl alcohol-water (2:1:1, i n an N H atmosphere). F o r detection, n i n h y d r i n a n d 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e w e r e u s e d . T h e content of n i n h y d r i n - p o s i t i v e p r o d u c t s i n hydrolysates of the i r r a d i a t e d samples of p o l y m y x i n w a s v e r y l o w i n c o m p a r i s o n w i t h t h e content of a m i n o acids f o r m i n g t h e o r i g i n a l p e p t i d e . F o r t h e i r i d e n t i f i c a tion a n d quantitative determination w e used quantitative paper chroma t o g r a p h y i n t h e m o d i f i c a t i o n of H e i l m a n et al. (6). T h i s m e t h o d w a s also u s e d f o r d e t e r m i n i n g the decrease of t h e f o u r b a s i c a m i n o acids of the p e p t i d e s t u d i e d . C o n s i d e r i n g t h e m a r k e d g r a d i n g of t h e r a d i a t i o n doses u s e d i n the present w o r k , t h e a t t a i n e d a c c u r a c y oi±5% can be considered as satisfactory. T h e r e c o m b i n a t i o n p r o d u c t s f o r m e d i n p o l y m y x i n solutions i r r a d i a t e d i n oxygen-free atmosphere w e r e separated f r o m t h e i r r a d i a t e d s o l u t i o n o n a c o l u m n of S e p h a d e x G 25 ( 5 0 X 1 c m . ) . T h e eluent w a s 0 . 0 5 M acetic a c i d f l o w i n g at t h e rate of 6 m l . / h r . R e c o m b i n a t i o n p r o d u c t s of h i g h e r m o l e c u l a r w e i g h t passed as t h e first f r a c t i o n . F r o m i r r a d i a t e d solutions of free d i a m i n o b u t y r i c a c i d a n d f r o m hydrolysates of i r r a d i a t e d p o l y m y x i n solutions, t h e r e c o m b i n a t i o n p r o d u c t s w e r e separated as the first f r a c t i o n o n a c o l u m n of S e p h a d e x G 10. 6 0

5

5

5

5

5

3


5

33.

KOPOLDOVA

Cyclic

Dodecapeptide

Polymyxin

475

B

6 - m e t h y l o c t a n o i c a c i d o c c u r r i n g i n the hydrolysates of p o l y m y x i n w a s q u a l i t a t i v e l y d e t e r m i n e d i n the f o r m of its m e t h y l ester b y means of gas c h r o m a t o g r a p h y ( A r g o n P y e instrument, 2 0 % R e o p l e x , C e l l i t e 545, 1 3 8 ° C , flow rate 20 m l . / m i n . ) . Results and

Discussion

Radiation Effects on Solutions of Free Diaminobutyric A c i d . A s has b e e n m e n t i o n e d earlier, a l o n g w i t h the s t u d y of p o l y m y x i n solutions w e Downloaded by UNIV OF GUELPH LIBRARY on June 18, 2012 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch033

h a v e also s t u d i e d r a d i a t i o n effects o n solutions of free d i a m i n o b u t y r i c a c i d , w h i c h w i t h its five m o l e c u l e s is c o n t a i n e d i n the o r i g i n a l p e p t i d e to a larger extent t h a n a n y other single c o m p o n e n t .

It c o u l d b e

expected

that the establishment of the m a i n m e c h a n i s m s of r a d i a t i o n d e g r a d a t i o n of free d i a m i n o b u t y r i c a c i d w i l l c o n t r i b u t e to the e l u c i d a t i o n of p o l y m y x i n radiolysis. It was f o u n d that the major m e c h a n i s m i n r a d i o l y s i s of d i a m i n o b u t y r i c a c i d i n o x y g e n a t e d s o l u t i o n is o x i d a t i v e d e a m i n a t i o n o n a-

or

y - p o s i t i o n . T h i s is clear f r o m a c o m p a r i s o n of the decrease i n the content of d i a m i n o b u t y r i c a c i d ( G / - M / 2 . 6 5 ) , w i t h the y i e l d of a m m o n i a ( G a n d w i t h the y i e l d s of d e a m i n a t e d p r o d u c t s ( S G «

2.0).

c a r b o n y l substances a n d k e t o a m i n o acids, /^-alanine a n d aspartic have

been

oxygen-free

found among atmosphere,

the

products

t w o of

the

2.1)

Aside from acid

of o x i d a t i v e d e a m i n a t i o n .

In

m a i n radiation mechanisms

reductive deamination ( G / - M / 1 . 4 and G N H

3

1.2)

are

and recombination

reactions l e a d i n g to p r o d u c t s of h i g h e r m o l e c u l a r w e i g h t . Radiation Effects on Solutions of Polymyxin. T h e decrease i n the content of p o l y m y x i n a n d its b i o l o g i c a l a c t i v i t y , as w e l l as the results of analyses f o r free a n d a m i d e - l i k e a m m o n i a , t o t a l content of c a r b o n y l sub stances, a n d the content of i n d i v i d u a l a m i n o acids i n the hydrolysates of i r r a d i a t e d samples are s u m m a r i z e d i n T a b l e I. Irradiation in an Oxygenated Solution. T h e l o w e s t dose of 1.36 10

5

X

r a d . d i d not p r o d u c e a n y significant changes i n the s o l u t i o n of p o l y

m y x i n . T h e content of the substance a n d its b i o l o g i c a l a c t i v i t y w e r e o n l y slightly reduced. O n i n c r e a s i n g the dose to 13.17

X

10

5

r a d , b o t h the content

p o l y m y x i n a n d its b i o l o g i c a l a c t i v i t y w e r e r e d u c e d b y a b o u t 44 to T h e d e c o m p o s i t i o n p r o d u c t was a p e p t i d e - l i k e substance w i t h

of

45%.

electro-

p h o r e t i c b e h a v i o r s i m i l a r to p o l y m y x i n , b u t it h a d a p o s i t i v e r e a c t i o n to 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e .

O n h y d r o l y z i n g the i r r a d i a t e d s o l u t i o n

w i t h 4N H C 1 at 9 5 ° C . , the substance was d e c o m p o s e d to a basic p e p t i d e , d i a m i n o b u t y r i c a c i d , a n d to a n u n i d e n t i f i e d k e t o a c i d .

A p p a r e n t l y , as a

result of i r r a d i a t i o n the p e p t i d e b o n d is a t t a c k e d a c c o r d i n g to Garrison's r e a c t i o n 3. F r o m the y i e l d of a m i d e - l i k e a m m o n i a , G 1.05,

it c a n


be

476

RADIATION CHEMISTRY

Table I.

Dose (rad)

?dium

0 r


Decrease in the Polymyxin B Content, Biological and Oxygen-free 0.5% Aqueous Solutions

Content of

pH

Polymyxin

6.2

1.36 X 10 »

13.17 X 10>

1

Oo N 2

+ 6.1

o

2

3.7

N

2

5.9

o

2

2.9

N

2

5.4

Biological Activity

3

Amidelike NH 3

Total

c=o

100%

100% 98% 98%

b

Free NH

97% 98%

+ +

+ +

+ +

55%

56%

0.4

1.2

2.0

56%

55%

0.1

0.4

0.15

—

1.2

4.8

5.7

—

0.4

1.6

0.35

—

—

4.4

13.6

—

—

1.2

3.6

r

66.10

131.10

1 b

—

5

q.

2.55

N

5.1

traces

9.3

3

2

1.25

Expressed in mmol./ml. or % of original. A plus sign ( + ) indicates a value which was not determined.

estimated that at this dose t h e extent of cleavage of t h e p e p t i d e b o n d is approximately 5 % . I r r a d i a t i o n i n o x y g e n a t e d solutions also leads to o x i d a t i v e d e a m i n a t i o n reactions o n some of the free - N H groups of d i a m i n o a c i d chains 2

a c c o r d i n g to R e a c t i o n S c h e m e 1. T h e s e reactions l e a d to the f o r m a t i o n of p e p t i d e s w h i c h c o n t a i n , i n a d d i t i o n to d i a m i n o b u t y r i c chains, d e a m i n a t e d derivatives w i t h a c a r b o n y l g r o u p . I n a strongly o x i d a t i v e m e d i u m these groups are f u r t h e r o x i d i z e d to - C O O H groups, as is w i t n e s s e d b y the presence of aspartic a c i d i n the hydrolysates of these peptides. T h e r e p l a c e m e n t i n t h e course of i r r a d i a t i o n of free - N H

2

groups b y c a r b o n y l

a n d c a r b o x y l groups leads to t h e f o r m a t i o n of n e u t r a l a n d a c i d i c p e p t i d e s , w h i c h w e r e d e t e c t e d i n solutions of p o l y m y x i n i r r a d i a t e d at doses of 66 X 1 0 r a d . a n d 131 X 10 r a d . T h e extent of cleavage of t h e p e p t i d e 5

r>

b o n d a c c o r d i n g to G a r r i s o n ' s r e a c t i o n increased to 1 0 % , a n d o n i r r a d i a t i o n at the highest dose i t f u r t h e r increased to 3 0 % . I n solutions of p o l y m y x i n i r r a d i a t e d at these doses, s m a l l amounts of free a m i n o acids h a v e b e e n f o u n d ( t h r e o n i n e , traces of l e u c i n e a n d p h e n y l a l a n i n e , a n d ^ - a l a n i n e ). T h e presence of these a m i n o acids c a n b e e x p l a i n e d b y the cleav age of p e p t i d e b o n d s a c c o r d i n g to R e a c t i o n 2. E v i d e n c e of this c a n b e


33.

KOPOLDOVA

Cyclic

Dodecapeptide

Polymyxin

477

B

A c t i v i t y , and Yield of Products Formed from Its Oxygenated Irradiated by the Doses 1.36 to 131.7 X 10 r a d 5

Decrease of Amino Acids (in hydrolyzates)

a

New Formed Amino Acids (in hydrolyzates)

di-NH butyric

Thr

Leu

Phe

Asp

fi-Ala

Gly, Se

butyric y-amino

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

0.3

traces

—

—

traces

—

—

0.25

1.2

0.5

traces

—

traces

—

—

0.75

2.2

0.6

0.1

—

0.1

—

—

1.5


2

2.3 (12%) 2.55 (10%)

0.7 (8%) 1.25 (15%)

0.8 (20%) 0.75 (18%)

1.25 (30%) 1.05 (25%)

6.4 (26%) 6.0 (24%)

3.1 (37%) 2.7 (32%)

3.1 (74%) 1.3 (32%)

4.1 (98%) 3.2 (78%)

10.0 (40%) 8.5 (34%)

6.7 (80%) 4.6 (55%)

4.15 (100%) 2.1 (50%)

4.2 (100%) 4.1 (98%)

A minus sign

( - ) indicates zero value.

seen i n the presence of ^ - a l a n i n e , w h i c h is p r o b a b l y f o r m e d b y

the

o x i d a t i o n of the a m i n o a l d e h y d e o b t a i n e d i n R e a c t i o n 2. I n a d d i t i o n to the above m e n t i o n e d o x i d a t i v e d e a m i n a t i o n reactions a n d Garrison's cleavage of the p e p t i d e b o n d , o x i d a t i o n a n d d e g r a d a t i o n reactions also take p l a c e i n the r e m a i n i n g a m i n o a c i d chains, i n c l u d i n g the benzene n u c l e u s of P h e , as c a n b e i n f e r r e d f r o m the presence of d i a l d e h y d e s w h i c h h a v e b e e n f o u n d i n i r r a d i a t e d solutions of p o l y m y x i n . A s a result of these reactions the content of i n d i v i d u a l a m i n o acids i n the hydrolysates of i r r a d i a t e d solutions is successively l o w e r , the decrease b e i n g largest f o r P h e , L e u , a n d for m e t h y l o c t a n o i c a c i d . Irradiation in Oxygen-Free Solutions of Polymyxin. dose d i d not p r o d u c e observable changes,

The

smallest

thus the results are s i m i l a r

to those o b t a i n e d w i t h o x y g e n a t e d solutions. O n i n c r e a s i n g the dose, the content of p o l y m y x i n a n d its b i o l o g i c a l a c t i v i t y w e r e r e d u c e d . O n the e l e c t r o p h o r e o g r a m of the i r r a d i a t e d sample w e h a v e f o u n d , aside f r o m the o r i g i n a l p e p t i d e , t w o r a d i a t i o n p r o d u c t s w i t h m a r k e d l y c h a n g e d c h r o m a t o g r a p h i c a n d electrophoretic

behavior.

O n e of these p r o d u c t s , w h i c h moves d o w n a c o l u m n of S e p h a d e x G 25


478

RADIATION CHEMISTRY

as the first f r a c t i o n , remains at the start of the e l e c t r o p h o r e o g r a m ;

1

appar

ently this is a h i g h e r m o l e c u l a r w e i g h t p r o d u c t f o r m e d b y a r e c o m b i n a tion reaction.

The

second

p r o d u c t is less basic

than

non-irradiated

polymyxin. I r r a d i a t i o n of p o l y m y x i n i n a n oxygen-free

atmosphere

at

higher

doses r e s u l t e d i n a n almost c o m p l e t e loss of b i o l o g i c a l a c t i v i t y ; the o r i g i n a l p e p t i d e c o u l d be d e t e r m i n e d o n l y i n trace a m o u n t .

H i g h e r amounts

of r a d i a t i o n p r o d u c t s d e s c r i b e d i n the p r e v i o u s section w e r e present.


the h y d r o l y s a t e of the i r r a d i a t e d s o l u t i o n , a 25 to 3 0 %

In

decrease was

d e t e r m i n e d for a l l a m i n o acids, except for p h e n y l a l a n i n e w h o s e decrease a m o u n t e d to 7 8 % . hydrolysates:

(1)

H o w e v e r , n e w a m i n o acids w e r e detected y-aminobutyric acid and

(2)

i n the

a-aminobutyric

acid.

B o t h of these substances h a v e b e e n f o u n d i n a n i r r a d i a t e d s o l u t i o n of d i a m i n o b u t y r i c a c i d w h e r e t h e y represent p r o d u c t s of r e d u c t i v e d e a m i n a t i o n reactions i n the y - a n d a-positions. T h e presence of y - a m i n o b u t y r i c a c i d i n the

h y d r o l y s a t e of i r r a d i a t e d p o l y m y x i n indicates

that i n a n

oxygen-free atmosphere r e d u c t i v e cleavage of the p e p t i d e b o n d a p p a r e n t l y takes p l a c e

between

some

of the

d i a m i n o b u t y r i c residues

(see

R e a c t i o n 7 ) . T h e presence of a - a m i n o b u t y r i c a c i d indicates that r e d u c t i v e d e a m i n a t i o n reactions

o c c u r i n i r r a d i a t e d s o l u t i o n of p o l y m y x i n ,

e x p l a i n i n g the f o r m a t i o n of the less basic p e p t i d e s w h i c h h a v e

thus been

d e t e c t e d i n the i r r a d i a t e d samples. I r r a d i a t i o n at the highest dose r e s u l t e d i n a n increase of the r e c o m b i n a t i o n p r o d u c t w h i c h remains at the start of the p a p e r

electrophoreo-

grams a n d c h r o m a t o g r a m s a n d gives a w e a k n i n h y d r i n - p o s i t i v e r e a c t i o n . It is s t r o n g l y

fluorescent

i n ultraviolet light.

F r o m the h y d r o l y s a t e of

the s a m p l e i r r a d i a t e d at the highest dose, w e have separated o n a c o l u m n of S e p h a d e x G 10 the first fractions.

T h e i r c h r o m a t o g r a p h i c a n d electro-

p h o r e t i c b e h a v i o r ( 7 ) c o r r e s p o n d e d w i t h that of the r e c o m b i n a t i o n p r o d ucts i s o l a t e d i n the same m a n n e r f r o m a n i r r a d i a t e d sample of free d i a m i n o b u t y r i c a c i d . It c a n b e c o n c l u d e d f r o m this that the m a i n p o r t i o n of r e c o m b i n a t i o n p r o d u c t s of p o l y m y x i n is f o r m e d as a result of r e c o m b i n a tion

between

d i a m i n o b u t y r i c chains.

H o w e v e r , other

reactions p r o b a b l y take place—e.g., reactions P h e , a n d L e u chains.

recombination

between diaminobutyric,

T h i s w o u l d e x p l a i n the m a r k e d decrease i n the

content of these substances. A d e t a i l e d analysis of these r e c o m b i n a t i o n p r o d u c t s has not b e e n c a r r i e d out. T h e results o u t l i n e d enable us to c o n c l u d e that w h e n the

dodeca

p e p t i d e is i r r a d i a t e d , it is subject to the same m e c h a n i s m s w h i c h h a v e been

established

for

i r r a d i a t e d solutions

of

simple peptides.

In

an

o x y g e n a t e d m e d i u m , r a d i a t i o n changes take p l a c e b o t h i n the side chains of a m i n o a c i d residues a n d i n the c y c l i c c h a i n of the p e p t i d e b o n d .


33.

KOPOLDOVA

Cyclic

Dodecapeptide

Polymyxin

Β

479

I n t h e first case o x i d a t i v e d e a m i n a t i o n reactions o c c u r o n t h e free N H groups of d i a m i n o b u t y r i c residues w i t h t h e f o r m a t i o n o f k e t o - a c y l peptides. D u r i n g f u r t h e r o x i d a t i o n , these substances are c h a n g e d i n t o a c i d peptides b y successive replacements of N H groups b y a l d e h y d i c a n d c a r b o x y l i c groups. T h e other a m i n o a c i d residues also u n d e r g o o x i d a t i o n a n d d e g r a d a t i o n reactions, w i t h the f o r m a t i o n of O H derivatives, f u r t h e r o x i d a t i o n p r o d u c t s o r s p l i t t i n g of t h e c a r b o n c h a i n , w h i c h is the case f o r P h e a n d L e u residues. T h e o r d e r of r a d i o s e n s i t i v i t y of these substances is i n agreement w i t h t h e results g i v e n b y A m b e , K u m t a , a n d T a p p e l ( I ) . O x i d a t i v e cleavage of - N H - C O - b o n d s a c c o r d i n g t o G a r r i s o n s m e c h a n i s m takes place s i m u l t a n e o u s l y w i t h r a d i a t i o n changes i n chains o f a m i n o acids residues. 2


2

I n a n oxygen-free m e d i u m these reactions are suppressed. A s w i t h r a d i a t i o n changes i n t h e p e p t i d e c h a i n , b o t h r e d u c t i v e a n d o x i d a t i v e cleavage o c c u r i n a n oxygen-free m e d i u m b u t to a lesser extent t h a n i n a n oxygenated m e d i u m . T h e m a i n reactions w h i c h o c c u r i n this m e d i u m are r e d u c t i v e d e a m i n a t i o n o f the free - N H groups of d i a m i n o b u t y r i c residues a n d r e c o m b i n a t i o n reactions w i t h t h e f o r m a t i o n o f h i g h e r m o l e c u l a r w e i g h t peptides. I t cannot b e e x c l u d e d that r e c o m b i n a t i o n reactions also take p l a c e b e t w e e n side chains o f t h e same p o l y m y x i n m o l e c u l e , w h i c h w o u l d l e a d to a d e f o r m a t i o n of its o r i g i n a l structure a n d t h e r e b y t o a change of its b e h a v i o r o n Sephadex. T o e l u c i d a t e t h e course of r e c o m b i n a t i o n reactions w o u l d r e q u i r e a m o r e d e t a i l e d analysis of t h e r e c o m b i n a t i o n p r o d u c t s . T h e results of this analysis c o u l d b e v e r y u s e f u l f o r a better u n d e r s t a n d i n g of r a d i a t i o n changes i n m o r e c o m p l e x peptides or proteins. 2

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

Ambe, K. S., Kumta, U. S., Tappel, A. L., Radiation Res. 15, 709 (1961). Conway, E. J., Berne,A.,Biochem. J. 27, 419 (1933). Garrison, W. M., Weeks, Β. M., Radiation Res. 17, 341 (1962). Goss, W.A.,Katz, E., Appl. Microbiol. 5, 95 (1957). Hatano, H.,J.Radiation Res. (Japan) 1, 38 (1960). Heilmann, J., Barrolier, J., Watzke, Ε., Z. Physiol Chem. 309, 219 (1957). Katrukha, G. S., Silaev, A. B., Kharzkhaeva, S. V.,Biokhimiya27, 549 (1962). Lappin, G. R., Clark, L.C.,Anal. Chem. 23, 541 (1951). Liebster, J.,Kopoldová,J., Radiation Res. 27, 162 (1966). Messer, M., Biochim. Biophys. Acta 17, 151 (1955). Řičieová, Α., Podojil, M., Folia Microbiologica 10, 299 (1965).

RECEIVED January 11, 1968.


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