15.5. 1976
The m o r t a l i t y w i t h p l a t i n u m , an i n e r t metal, was low a n d m i g h t be due to t h e t o x i c i t y of t h e s a l t (BaC12) or to HC1 a n d HOC1 f o r m e d as a r e s u l t of r e a c t i o n b e t w e e n chlorine a n d w a t e r molecules. The h i g h e s t m o r t a l i t y w i t h 2 C1- -> CIe + 2e C12 q- H20 = HC1 + HOC1 BaCI~ (Figure 3) m i g h t be due to t h e t o x i c i t y of b a r i u m because t h e c o n t r o l w i t h BaCI~ alone s h o w e d 18% m o r tality. 80
573
Specialia
%
DPt.electrode
70
I Cu.electrode 0 Cu.electrode (N-pole at cathode (N-poleat anode)
~ Cu.electrode
60 50
The a v e r a g e p e r c e n t a g e m o r t a l i t y u n d e r crossed fields (E H) was t h e s a m e as u n d e r E H . N + a n d w o u l d t h e r e fore s t a n d for b o t h t h e s i t u a t i o n s in F i g u r e 3. I n solutions of f e r r o - m a g n e t i c salts t h e r e was a significant increase (at 0.05 level) in n e m a t o d e m o r t a l i t y ill b o t h electric a n d m a g n e t i c fields (E H, E H.N+). I n CoCI~ t h e r e was a significant decrease (at 0.05 level) in m o r t a l i t y u n d e r E H . N - . No n e m a t o d e s d i e d in t h e c o n t r o l of 4. The r a t e of c h e m i c a l r e a c t i o n is a l t e r e d u n d e r t h e influence of m a g n e t i c fields TM. BHATNAGAR a n d MATHUR 9 s u g g e s t e d t h a t t h e a l t e r e d r a t e s in a n u m b e r of c h e m i c a l r e a c t i o n s were caused b y a s t i r r i n g effect of t h e m a g n e t i c field on t h e solution as well as a n a l i g n m e n t of p a r a m a g n e t i c m o m e n t s of t h e molecules involved. I n t h e p r e s e n t e x p e r i m e n t b i o m a g n e t i c effects were o b s e r v e d o n l y in case of I e r r o - m a g n e t i c salts and, as such, could b e ascribed to t h e a l i g n m e n t of para-magnetic m o m e n t s of t h e molecules w h i c h m i g h t h a v e i n c r e a s e d t h e v e l o c i t y of electroc h e m i c a l reactions.
40
30 ::
20 u
10
~ c
:: b i
bb b
bb
b
aaa
11
FeC, CoCI2 NiClz MnC[2 CrCl$ ZnOt2 BaC[2 NaG[
KCI
Fig. 3. Nematode mortality resulting from electrolysis of 9 salt solutions at 5• 10-a g lit-1 concentration for 2 h using Cu- and Ptelectrodes with and without magnetic field. Controls for each electrolyte excepting BaCI~: 1. without electric and magnetic field - no mortality in 2 h; 2. with magnetic field alone - no mortality in 2 h. 18% mortality in BaC12 in both 1 and 2. a, b, e, d, different letters indicate significant difference in mortality at 0.01 level within the graph for an electrolyte.
Degradation
of P h e n y l a l a n i n e
9 S. S. BHATNAGARand K. N. MATHUR, Physical Principles and Applications of Magneto-Chemistry (Macmillan and Co., London 1935), p. 327. 10 I. L. MIJLAY and L. N. MULAY,in Biological E[]ects o] Magnetic Fields (Ed. M. F. BAR~OTHu Plenum Press, New York 1964), p. 146. 1I I. L. SILVER and C. A. TOBIAS, in Space Radiation Biology and Related Topics (Eds. C. A~ TOBIAS and P. TODD; Academic Press, New York 1974), p. 293. 1~ E. BARNES, P h . D . Thesis, Biology Department, University of Houston (1967).
in t h e P r e s e n c e of H y d r o g e n
Peroxide
A. S. ANSARI, S. TAHIB a n d RASHID ALll
Department o/Biochemistry, Faculty o/Medicine, d ligarh Muslim University, A ligarh 202 O01 (India), 18 November 1975. Summary. U V - i r r a d i a t i o n of p h e n y l a l a n i n e b y 253.7 n m light in t h e p r e s e n c e of h y d r o g e n p e r o x i d e f o r m e d 5 n i n h y d r i n r e a c t i v e p r o d u c t s a n d a m m o n i a . F o u r of t h e m were i d e n t i f i e d as a s p a r t i c acid, serine, alanine a n d lysine. Stable a n d u n s t a b l e s u b s t a n c e s w h i c h p r o d u c e c h e m i c a l effects h a v e b e e n s h o w n t o be f o r m e d b y u l t r a v i o l e t (UV) light. One s u c h s u b s t a n c e is h y d r o g e n p e r o x i d e f o r m e d d u r i n g U V - i r r a d i a t i o n of n i c o t i n e in t h e p r e s e n c e of m e t h y l e n e blue 2. EERRARI a n d PASSERA 3 h a v e s h o w n t h e f o r m a t i o n of serine b y U V - i r r a d i a t i o n of a s p a r t i c acid. T h e y s u g g e s t e d t h a t t h e h y d r o x y l radical for its f o r m a t i o n c o m e s f r o m h y d r o g e n peroxide, p r o d u c e d b y U V a c t i o n on o x y g e n p r e s e n t in solutions. W e n o t i c e d d i f f e r e n t b e h a v i o u r of h y d r o g e n p e r o x i d e on U V - i r r a d i a t i o n . E x p o s u r e t o 253.7 n m light results in its d i s a p p e a r a n c e a n d t h e s a m e t r e n d was n o t i c e d w h e n i r r a d i a t e d in t h e p r e s e n c e of citrulline a n d arginine. H o w e v e r , in t h e p r e s e n c e of Iysine, t y r o s i n e a n d p h e n y l alanine, t h e a m o u n t r e m a i n e d u n c h a n g e d w i t h v a r y i n g r a d i a t i o n doses. I n v i e w of t h e s e o b s e r v a t i o n s , a n inv e s t i g a t i o n of t h e effect of 253.7 nlI1 l i g h t on a q u e o u s solutions of p h e n y l a l a n i n e in t h e p r e s e n c e of h y d r o g e n
p e r o x i d e was u n d e r t a k e n . Special e m p h a s i s has b e e n laid on t h e s e p a r a t i o n a n d i d e n t i f i c a t i o n of a m i n o acids t h u s formed. A s h o r t w a v e l e n g t h U V - l a m p S p e c t r o l i n e (manufact u r e d b y B l a c k L i g h t E a s t e r n , Inc. U S A , m o d e l R-51) h a v i n g m a x i m u m emission a t 253.7 n m a n d i n t e n s i t y 155 ~w/cm 2 a t a d i s t a n c e of 18" was e m p l o y e d as r a d i a t i o n source. A q u e o u s solution of 2 m M p h e n y l a l a n i n e were e x p o s e d t o U V as p r e v i o u s l y r e p o r t e d 4. To s t u d y t h e effects of h y d r o g e n peroxide, e q u a l a m o u n t s (v/v) were 1 Acknowledgments. This work was supported by the Council of Scientific and Industrial Research. Thanks are due to the Head of the Department for facilities and to Mr. RAKESHC. SHARMAfor help. L. WEIL and J: MA~ER, Arch. Biochem. Biophys. 29, 241 (1950). 8 G. FERRARIand C. PASSERA,Photochem. Photobiol. 7, 155 (1962). 4 M. FAHIM and RASmD ALl, Indian. J. Biochem. 6, 232 (1959).
574
Speeialia
Formation of amino acids on UV-irradiation (28.28 • 107 ergs/cm~) of phenylatanine in the presence of hydrogen peroxide Amino acid
Peak number (Fig. 1)
Amount (btmoles)
Aspartic acid Serine Alanine Lysine
1 2 3 5
84 136 159 30
O,7
2
O.E
(1[
o* 0/ g
0,2
; f ....
0.1
% 20 40 I,
80
Too 12o 1~o ;"2~o 240 26o 28o 34s
9 I. pH4.2,50~ --- pH 4.2.75~ Fractionnumber "--"lpH8.3~ pH9.2,25~-"r pH 11-'1
oH3.4,37.5~
Fig. 1. Ion exchange chromatographic profile of UV-irradiated (28.28 • 103 ergs/cm2) phenylalanine in the presence of hydrogen peroxide.
80
/
0
,
,-,
-0
5
10
i5
r
i
20
o
25
30
Radiation dose (xl0-7ergs/cm2)
Fig. 2. Destruction of phenylalanine on UV-irradiation in the presence (0--0) and absence (O-- 9 of hydrogen peroxide.
16
1.2
E
"~ 0.8
04
O0
o
5
10
15
Radiation dose(•
o
20
EXPERIENTIA 32/5
a d d e d t o t h e a m i n o acid solution. T h e a c t u a l a m o u n t of h y d r o g e n p e r o x i d e was e s t i m a t e d b y i o d o m e t r y 5, its conc e n t r a t i o n was m a i n t a i n e d a t 1.87 m g / m l unless o t h e r w i s e m e n t i o n e d . P r o p e r c o n t r o l s were i n c l u d e d in e a c h set of experiments. Descending paper chromatography on W h a t m a n No. 1 p a p e r was e m p l o y e d for s e p a r a t i o n of p h e n y l a l a n i n e a n d its U V d e g r a d a t i o n p r o d u c t u s i n g nb u t a n o l / a c e t i c a c i d / w a t e r (60: 15:25) as t h e d e v e l o p i n g solvent. I o n e x c h a n g e c h r o m a t o g r a p h y on D o w e x 50*W-X8 resin was t h e basic m e t h o d used in t h e s e p a r a t i o n of a m i n o acids e s s e n t i a l l y b y t h e p r o c e d u r e of MOORE a n d STEIN 6. T h e s e p a r a t i o n was c a r r i e d o u t on a 0.9 • 100 c m j a c k e t e d c o l u m n w i t h t h e s e q u e n c e ,of buffers a n d t e m p e r a t u r e as s h o w n in F i g u r e 1. I n e a c h of t h e 345 fractions, 2.0 m l were collected a t t h e r a t e of 1.0 m l / 6 min. T h e a m o u n t of a m i n o acids was e s t i m a t e d b y modified n i n h y d r i n r e a g e n t ~. To c o n f i r m t h e i d e n t i t i e s of t h e a m i n o acids formed, d i n i t r o p h e n o l (DNP) d e r i v a t i v e s were p r e p a r e d e s s e n t i a l l y b y SANGER'S m e t h o d s a n d were s e p a r a t e d b y p a p e r chrom a t o g r a p h y u s i n g p y r i d i n e / i s o a m y l alcohol/1.6 N a m m o n i a (6: 14: 20) as t h e d e v e l o p i n g s o l v e n t . D N P d e r i v a t i v e s of reference a m i n o acids were p r e p a r e d b y t h e s a m e procedure. T h e f o r m a t i o n of a m m o n i a was e s t i m a t e d b y Nessler's r e a g e n t as p r e v i o u s l y reportedg. U V - i r r a d i a t e d p h e n y l a l a n i n e in t h e p r e s e n c e of h y d r o gen p e r o x i d e ( 2 8 . 2 8 • e r g s / c m 2) g a v e 5 n i n h y d r i n r e a c t i v e s p o t s of R f v a l u e s 0.16, 0.25, 0.30, 0.42 a n d 0.52 besides p h e n y l a l a n i n e (Rf 0.63). W h e n t h e i r r a d i a t e d s a m p l e s s u b j e c t e d to ion e x c h a n g e c h r o m a t o g r a p h y , 5 d i s t i n c t p e a k s were o b t a i n e d (Figure 1). 4 p e a k s could be identified o n t h e basis of e l u t i o n v o l u m e as well as b y preparing their DNP derivatives, running authentic DNP a m i n o acids as s t a n d a r d s . T h e p r o d u c t s identified are a s p a r t i c acids, serine, a l a n i n e a n d lysine c o r r e s p o n d i n g to p e a k s No. 1, 2, 3 a n d 5 respectively. P e a k No. 4 was n o t identified. Alanine, serine a n d a s p a r t i c acid are f o r m e d in a p p r e c i a b l e a m o u n t s , while lysine a c c o u n t s for o n l y a s m a l l f r a c t i o n (Table). E s t i m a t i o n of p h e n y l a l a n i n e b y n i n h y d r i n r e a g e n t r e v e a l e d t h a t t h e d e s t r u c t i o n of a m i n o acid was several fold w h e n i r r a d i a t e d in t h e p r e s e n c e of h y d r o g e n p e r o x i d e (Figure 2). I r r a d i a t i o n w i t h o u t h y d r o gen p e r o x i d e was a l m o s t ineffective. A m m o n i a was t h e o t h e r p r o d u c t f o r m e d as a r e s u l t of U V - i r r a d i a t i o n of p h e n y l a l a n i n e , its r a t e of f o r m a t i o n i n c r e a s i n g c o n s i d e r a b l y w h e n h y d r o g e n p r o x i d e was a d d e d (Figure 3). T h e m e c h a n i s m s u s p e c t e d t o b e i n v o l v e d in t h e p h o t o d e g r a d a t i o n of citrulline a n d a r g i n i n e in t h e p r e s e n c e of h y d r o g e n p e r o x i d e seems to be d i f f e r e n t f r o m t h e m e c h a n i s m o p e r a t i n g in t h e d e g r a d a t i o n of p h e n y l a l a n i n e . I n t h i s case, t h e e x c i t e d h y d r o g e n p e r o x i d e m o l e c u l e t r a n s f e r s a m a j o r p o r t i o n of its a b s o r b e d e n e r g y t o t h e s u b s t r a t e w h i c h m a y cleave a n u m b e r of b o n d s , p r o d u c i n g s e v e r a l r a d i c a l s a n d t h u s f o r m i n g a n u m b e r of p r o d u c t s . If a n y p a r t of t h e e n e r g y is r e t a i n e d , t h i s is n o t sufficient to p h o t o l y z e t h e h y d r o g e n p e r o x i d e molecule. T h i s is t h e r e a s o n w h y d u r i n g p h o t o l y s i s of p h e n y l a l a n i n e , h y d r o g e n p e r o x i d e was n o t c o n s u m e d a t all, i r r e s p e c t i v e t o its a m o u n t a n d r a d i a t i o n dose. T h e f o r m a t i o n of a l a n i n e a n d serine m i g h t b e d u e t o t h e p a r t i c i p a t i o n of t h e a l a n i n e side chain, while lysine a n d a s p a r t i c acid seem t o b e p r o d u c t s of r i n g r u p t u r e .
o
25
30 2)
Fig. 3. Formation of ammonia from phenylalanine on UV-irradiation in the presence (Q--O) and absence (0-- 9 of hydrogen peroxide.
A. I. VOGEL, in A Text Book o] Quantitative Inorganic Analysis (John Wiley and Sons Inc., New York 1961, p. 363. S. MOORE and W. H. STEIX, J. biol. Chem. 192, 663 (1951). 7 S. MOORE and W. H. STEIN, J. biol. Chem. 211, 907 (1954). s F. SANGER, Bioehem. J. 39, 507 (1945). 9 M. ASIF, RASHID ALI and I. HOSAIN, Indian J. Biochem. 6, 90 (1969).
The m o r t a l i t y w i t h p l a t i n u m , an i n e r t metal, was low a n d m i g h t be due to t h e t o x i c i t y of t h e s a l t (BaC12) or to HC1 a n d HOC1 f o r m e d as a r e s u l t of r e a c t i o n b e t w e e n chlorine a n d w a t e r molecules. The h i g h e s t m o r t a l i t y w i t h 2 C1- -> CIe + 2e C12 q- H20 = HC1 + HOC1 BaCI~ (Figure 3) m i g h t be due to t h e t o x i c i t y of b a r i u m because t h e c o n t r o l w i t h BaCI~ alone s h o w e d 18% m o r tality. 80
573
Specialia
%
DPt.electrode
70
I Cu.electrode 0 Cu.electrode (N-pole at cathode (N-poleat anode)
~ Cu.electrode
60 50
The a v e r a g e p e r c e n t a g e m o r t a l i t y u n d e r crossed fields (E H) was t h e s a m e as u n d e r E H . N + a n d w o u l d t h e r e fore s t a n d for b o t h t h e s i t u a t i o n s in F i g u r e 3. I n solutions of f e r r o - m a g n e t i c salts t h e r e was a significant increase (at 0.05 level) in n e m a t o d e m o r t a l i t y ill b o t h electric a n d m a g n e t i c fields (E H, E H.N+). I n CoCI~ t h e r e was a significant decrease (at 0.05 level) in m o r t a l i t y u n d e r E H . N - . No n e m a t o d e s d i e d in t h e c o n t r o l of 4. The r a t e of c h e m i c a l r e a c t i o n is a l t e r e d u n d e r t h e influence of m a g n e t i c fields TM. BHATNAGAR a n d MATHUR 9 s u g g e s t e d t h a t t h e a l t e r e d r a t e s in a n u m b e r of c h e m i c a l r e a c t i o n s were caused b y a s t i r r i n g effect of t h e m a g n e t i c field on t h e solution as well as a n a l i g n m e n t of p a r a m a g n e t i c m o m e n t s of t h e molecules involved. I n t h e p r e s e n t e x p e r i m e n t b i o m a g n e t i c effects were o b s e r v e d o n l y in case of I e r r o - m a g n e t i c salts and, as such, could b e ascribed to t h e a l i g n m e n t of para-magnetic m o m e n t s of t h e molecules w h i c h m i g h t h a v e i n c r e a s e d t h e v e l o c i t y of electroc h e m i c a l reactions.
40
30 ::
20 u
10
~ c
:: b i
bb b
bb
b
aaa
11
FeC, CoCI2 NiClz MnC[2 CrCl$ ZnOt2 BaC[2 NaG[
KCI
Fig. 3. Nematode mortality resulting from electrolysis of 9 salt solutions at 5• 10-a g lit-1 concentration for 2 h using Cu- and Ptelectrodes with and without magnetic field. Controls for each electrolyte excepting BaCI~: 1. without electric and magnetic field - no mortality in 2 h; 2. with magnetic field alone - no mortality in 2 h. 18% mortality in BaC12 in both 1 and 2. a, b, e, d, different letters indicate significant difference in mortality at 0.01 level within the graph for an electrolyte.
Degradation
of P h e n y l a l a n i n e
9 S. S. BHATNAGARand K. N. MATHUR, Physical Principles and Applications of Magneto-Chemistry (Macmillan and Co., London 1935), p. 327. 10 I. L. MIJLAY and L. N. MULAY,in Biological E[]ects o] Magnetic Fields (Ed. M. F. BAR~OTHu Plenum Press, New York 1964), p. 146. 1I I. L. SILVER and C. A. TOBIAS, in Space Radiation Biology and Related Topics (Eds. C. A~ TOBIAS and P. TODD; Academic Press, New York 1974), p. 293. 1~ E. BARNES, P h . D . Thesis, Biology Department, University of Houston (1967).
in t h e P r e s e n c e of H y d r o g e n
Peroxide
A. S. ANSARI, S. TAHIB a n d RASHID ALll
Department o/Biochemistry, Faculty o/Medicine, d ligarh Muslim University, A ligarh 202 O01 (India), 18 November 1975. Summary. U V - i r r a d i a t i o n of p h e n y l a l a n i n e b y 253.7 n m light in t h e p r e s e n c e of h y d r o g e n p e r o x i d e f o r m e d 5 n i n h y d r i n r e a c t i v e p r o d u c t s a n d a m m o n i a . F o u r of t h e m were i d e n t i f i e d as a s p a r t i c acid, serine, alanine a n d lysine. Stable a n d u n s t a b l e s u b s t a n c e s w h i c h p r o d u c e c h e m i c a l effects h a v e b e e n s h o w n t o be f o r m e d b y u l t r a v i o l e t (UV) light. One s u c h s u b s t a n c e is h y d r o g e n p e r o x i d e f o r m e d d u r i n g U V - i r r a d i a t i o n of n i c o t i n e in t h e p r e s e n c e of m e t h y l e n e blue 2. EERRARI a n d PASSERA 3 h a v e s h o w n t h e f o r m a t i o n of serine b y U V - i r r a d i a t i o n of a s p a r t i c acid. T h e y s u g g e s t e d t h a t t h e h y d r o x y l radical for its f o r m a t i o n c o m e s f r o m h y d r o g e n peroxide, p r o d u c e d b y U V a c t i o n on o x y g e n p r e s e n t in solutions. W e n o t i c e d d i f f e r e n t b e h a v i o u r of h y d r o g e n p e r o x i d e on U V - i r r a d i a t i o n . E x p o s u r e t o 253.7 n m light results in its d i s a p p e a r a n c e a n d t h e s a m e t r e n d was n o t i c e d w h e n i r r a d i a t e d in t h e p r e s e n c e of citrulline a n d arginine. H o w e v e r , in t h e p r e s e n c e of Iysine, t y r o s i n e a n d p h e n y l alanine, t h e a m o u n t r e m a i n e d u n c h a n g e d w i t h v a r y i n g r a d i a t i o n doses. I n v i e w of t h e s e o b s e r v a t i o n s , a n inv e s t i g a t i o n of t h e effect of 253.7 nlI1 l i g h t on a q u e o u s solutions of p h e n y l a l a n i n e in t h e p r e s e n c e of h y d r o g e n
p e r o x i d e was u n d e r t a k e n . Special e m p h a s i s has b e e n laid on t h e s e p a r a t i o n a n d i d e n t i f i c a t i o n of a m i n o acids t h u s formed. A s h o r t w a v e l e n g t h U V - l a m p S p e c t r o l i n e (manufact u r e d b y B l a c k L i g h t E a s t e r n , Inc. U S A , m o d e l R-51) h a v i n g m a x i m u m emission a t 253.7 n m a n d i n t e n s i t y 155 ~w/cm 2 a t a d i s t a n c e of 18" was e m p l o y e d as r a d i a t i o n source. A q u e o u s solution of 2 m M p h e n y l a l a n i n e were e x p o s e d t o U V as p r e v i o u s l y r e p o r t e d 4. To s t u d y t h e effects of h y d r o g e n peroxide, e q u a l a m o u n t s (v/v) were 1 Acknowledgments. This work was supported by the Council of Scientific and Industrial Research. Thanks are due to the Head of the Department for facilities and to Mr. RAKESHC. SHARMAfor help. L. WEIL and J: MA~ER, Arch. Biochem. Biophys. 29, 241 (1950). 8 G. FERRARIand C. PASSERA,Photochem. Photobiol. 7, 155 (1962). 4 M. FAHIM and RASmD ALl, Indian. J. Biochem. 6, 232 (1959).
574
Speeialia
Formation of amino acids on UV-irradiation (28.28 • 107 ergs/cm~) of phenylatanine in the presence of hydrogen peroxide Amino acid
Peak number (Fig. 1)
Amount (btmoles)
Aspartic acid Serine Alanine Lysine
1 2 3 5
84 136 159 30
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Too 12o 1~o ;"2~o 240 26o 28o 34s
9 I. pH4.2,50~ --- pH 4.2.75~ Fractionnumber "--"lpH8.3~ pH9.2,25~-"r pH 11-'1
oH3.4,37.5~
Fig. 1. Ion exchange chromatographic profile of UV-irradiated (28.28 • 103 ergs/cm2) phenylalanine in the presence of hydrogen peroxide.
80
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,
,-,
-0
5
10
i5
r
i
20
o
25
30
Radiation dose (xl0-7ergs/cm2)
Fig. 2. Destruction of phenylalanine on UV-irradiation in the presence (0--0) and absence (O-- 9 of hydrogen peroxide.
16
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"~ 0.8
04
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Radiation dose(•
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EXPERIENTIA 32/5
a d d e d t o t h e a m i n o acid solution. T h e a c t u a l a m o u n t of h y d r o g e n p e r o x i d e was e s t i m a t e d b y i o d o m e t r y 5, its conc e n t r a t i o n was m a i n t a i n e d a t 1.87 m g / m l unless o t h e r w i s e m e n t i o n e d . P r o p e r c o n t r o l s were i n c l u d e d in e a c h set of experiments. Descending paper chromatography on W h a t m a n No. 1 p a p e r was e m p l o y e d for s e p a r a t i o n of p h e n y l a l a n i n e a n d its U V d e g r a d a t i o n p r o d u c t u s i n g nb u t a n o l / a c e t i c a c i d / w a t e r (60: 15:25) as t h e d e v e l o p i n g solvent. I o n e x c h a n g e c h r o m a t o g r a p h y on D o w e x 50*W-X8 resin was t h e basic m e t h o d used in t h e s e p a r a t i o n of a m i n o acids e s s e n t i a l l y b y t h e p r o c e d u r e of MOORE a n d STEIN 6. T h e s e p a r a t i o n was c a r r i e d o u t on a 0.9 • 100 c m j a c k e t e d c o l u m n w i t h t h e s e q u e n c e ,of buffers a n d t e m p e r a t u r e as s h o w n in F i g u r e 1. I n e a c h of t h e 345 fractions, 2.0 m l were collected a t t h e r a t e of 1.0 m l / 6 min. T h e a m o u n t of a m i n o acids was e s t i m a t e d b y modified n i n h y d r i n r e a g e n t ~. To c o n f i r m t h e i d e n t i t i e s of t h e a m i n o acids formed, d i n i t r o p h e n o l (DNP) d e r i v a t i v e s were p r e p a r e d e s s e n t i a l l y b y SANGER'S m e t h o d s a n d were s e p a r a t e d b y p a p e r chrom a t o g r a p h y u s i n g p y r i d i n e / i s o a m y l alcohol/1.6 N a m m o n i a (6: 14: 20) as t h e d e v e l o p i n g s o l v e n t . D N P d e r i v a t i v e s of reference a m i n o acids were p r e p a r e d b y t h e s a m e procedure. T h e f o r m a t i o n of a m m o n i a was e s t i m a t e d b y Nessler's r e a g e n t as p r e v i o u s l y reportedg. U V - i r r a d i a t e d p h e n y l a l a n i n e in t h e p r e s e n c e of h y d r o gen p e r o x i d e ( 2 8 . 2 8 • e r g s / c m 2) g a v e 5 n i n h y d r i n r e a c t i v e s p o t s of R f v a l u e s 0.16, 0.25, 0.30, 0.42 a n d 0.52 besides p h e n y l a l a n i n e (Rf 0.63). W h e n t h e i r r a d i a t e d s a m p l e s s u b j e c t e d to ion e x c h a n g e c h r o m a t o g r a p h y , 5 d i s t i n c t p e a k s were o b t a i n e d (Figure 1). 4 p e a k s could be identified o n t h e basis of e l u t i o n v o l u m e as well as b y preparing their DNP derivatives, running authentic DNP a m i n o acids as s t a n d a r d s . T h e p r o d u c t s identified are a s p a r t i c acids, serine, a l a n i n e a n d lysine c o r r e s p o n d i n g to p e a k s No. 1, 2, 3 a n d 5 respectively. P e a k No. 4 was n o t identified. Alanine, serine a n d a s p a r t i c acid are f o r m e d in a p p r e c i a b l e a m o u n t s , while lysine a c c o u n t s for o n l y a s m a l l f r a c t i o n (Table). E s t i m a t i o n of p h e n y l a l a n i n e b y n i n h y d r i n r e a g e n t r e v e a l e d t h a t t h e d e s t r u c t i o n of a m i n o acid was several fold w h e n i r r a d i a t e d in t h e p r e s e n c e of h y d r o g e n p e r o x i d e (Figure 2). I r r a d i a t i o n w i t h o u t h y d r o gen p e r o x i d e was a l m o s t ineffective. A m m o n i a was t h e o t h e r p r o d u c t f o r m e d as a r e s u l t of U V - i r r a d i a t i o n of p h e n y l a l a n i n e , its r a t e of f o r m a t i o n i n c r e a s i n g c o n s i d e r a b l y w h e n h y d r o g e n p r o x i d e was a d d e d (Figure 3). T h e m e c h a n i s m s u s p e c t e d t o b e i n v o l v e d in t h e p h o t o d e g r a d a t i o n of citrulline a n d a r g i n i n e in t h e p r e s e n c e of h y d r o g e n p e r o x i d e seems to be d i f f e r e n t f r o m t h e m e c h a n i s m o p e r a t i n g in t h e d e g r a d a t i o n of p h e n y l a l a n i n e . I n t h i s case, t h e e x c i t e d h y d r o g e n p e r o x i d e m o l e c u l e t r a n s f e r s a m a j o r p o r t i o n of its a b s o r b e d e n e r g y t o t h e s u b s t r a t e w h i c h m a y cleave a n u m b e r of b o n d s , p r o d u c i n g s e v e r a l r a d i c a l s a n d t h u s f o r m i n g a n u m b e r of p r o d u c t s . If a n y p a r t of t h e e n e r g y is r e t a i n e d , t h i s is n o t sufficient to p h o t o l y z e t h e h y d r o g e n p e r o x i d e molecule. T h i s is t h e r e a s o n w h y d u r i n g p h o t o l y s i s of p h e n y l a l a n i n e , h y d r o g e n p e r o x i d e was n o t c o n s u m e d a t all, i r r e s p e c t i v e t o its a m o u n t a n d r a d i a t i o n dose. T h e f o r m a t i o n of a l a n i n e a n d serine m i g h t b e d u e t o t h e p a r t i c i p a t i o n of t h e a l a n i n e side chain, while lysine a n d a s p a r t i c acid seem t o b e p r o d u c t s of r i n g r u p t u r e .
o
25
30 2)
Fig. 3. Formation of ammonia from phenylalanine on UV-irradiation in the presence (Q--O) and absence (0-- 9 of hydrogen peroxide.
A. I. VOGEL, in A Text Book o] Quantitative Inorganic Analysis (John Wiley and Sons Inc., New York 1961, p. 363. S. MOORE and W. H. STEIX, J. biol. Chem. 192, 663 (1951). 7 S. MOORE and W. H. STEIN, J. biol. Chem. 211, 907 (1954). s F. SANGER, Bioehem. J. 39, 507 (1945). 9 M. ASIF, RASHID ALI and I. HOSAIN, Indian J. Biochem. 6, 90 (1969).
