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Oxidation of ascorbic acid by terramycin
SCORBIC
ACID
BY TERRA1
by ANI
ANn C. R. K R I S H N A M U R T I
lral Drug Research Institute, Lucknow, (India)
While s t u d y i n g t h e effect of a n t i b i o t i c s on t h e c y~tochrome tocl ox m s u a l l y h i g h e n d o g e n o u s o x y g e n u p t a k e w a s o b s e r v e d in con ne C, t e r r a m y c i n a n d a s c o r b i c a c i d w e r e i n c u b a t e d tt,o g e t h e r i~ iae. T h i s e n h a n c e d o x y g e n c o n s u m p t i o n w a s subseqt u e n t l y e, e o x i d a t i o n of a s c o r b i c a c i d b y t e r r a m y c i n t o t h e d e h y d r o fi r t a n c e of a s c o r b i c a c i d in t h e m a i n t e n a n c e of ooxid~ xidation red gical s y s t e m s ~, it w a s c o n s i d e r e d of i n t e r e s t to s t u d y in s o ~ r e d in t h i s i n t e r e s t i n g c a t a l y t i c p r o p e r t y of t h e aanti~ ntibiotic.
,,f h e a r t 1, [ch c y t o :e of t h e be due w of t h e n t i a l s of factors
EXPERIMENTAL
Antibiotics and other chemicals
['he following crystalline antibiotic obtained through the courtesy co The of their respective m man anufacturers ers were used in this study. Penicillin G (Indian Penicillin Committee), C Dihydrostreptomyc rcin zte (Glaxo Laboratories), Aureomycin hydrochloride (Lederl sulphate Lederle Laboratories), Chloramphenic ~henicol (Parkee Davis & Company), Neomycin sulphate (Upjohn & Compan Lpany) and Terramycin hydrochlori~ rochloride (Charles Pfizer & Company). Ascorbic ~scorbic acid and other chemicals employed were of reage~ gent grade purchased from Britifish Drug Houses Ltd., Charles Pfizer or Nutritional Bio-chemicals. Manometry
Conventional Warburg techniques es were adopted using flasks with centre cups and single side arn= ~rms. Analytical method
Ascorbic acid was estimatedfl according to ROE AND OESTERLING3. Spot tests for copper in the dru ]rugs used were carried out with dithiozonc reagent 4 (io mg in too ml T chloroform). Antibiotic assay
Terramycin was tested by the :he standard cup assay method ~ with S. aureus (Oxford) and E. ec cell as test organisms. Fluorimetry
The fluorescence of terramy 'cin was measured in a Lumetron Fluorimetcr 4o2 E using the tilt filter accessories for riboflavin. * An abstract of this paper was as :presented at a symposium on "Antibacterial substances from soil, so plants and other sources" held under the auspices of the Pharmaceuticals and Drugs Committe9e~ Council of Scientific and Industrial ial Research, at Bombay on the 26th March I953. Re/erenees p. 509.
RESULTS
(m ascorbic acid d to pH 7 were tipped in from the phate buffer pH 7.o and I ml ot were taken over a period of one he various reacting systems in 6o
the flask solution ) mimlte given ill
TABLE I F ASCORBIC ACID IN P R E S E N C E
A ntibiotic added
(Control) Aureomycin hydrochloride Chloramphenicol Neomycin sulphate Dihydrostreptomycin sulphatc Penicillin G Terramycin hydrochloride Water
O x. y. g .~ -. c .~ d. . microliters
25.0 24.2 25.7 21. 5 30.0 18.6 168.4
OF 6 ANt
- - ~ o / o g . , , ~ , , .... or inhibition
v+. -+
3.: 2.~ i4., 2o.i 25.q 573 -*
ill the antibiotics were found to affect the oxidation one ( way ( . Aureol, neomycin and penicilhn inhibited the oxidation, the maximui g shown nicillin G. Dihydrostreptomycin and terramycin accelerated acce tile rate ot OXldatloi tt :idation. atalytic action of terramycin was most pronounced, subsequent work was, therether( fore, confined to this antibiotic.
qcily o/the reaction with respect to substrate Specificity simi action on other reducin g Inn order to find out whether terramycin exerted a similar subst~ were tested: agentss and chemicals of biological interest following substances ~lucose, gluconic acid, lactic acid, pyruvic acid, sorbitol, sorbose, quercitol, glutathion tathione, Glucose le hydrochloride, sodium bisulphite, sodium thiosulphate, phenylenediamine, p sodium nitrite, nitrit cysteine din, cholesterol and vegetable lecithin (all these substances in I mg/ml adjusted to pH 7.0 adrenalin 7.0) ; -,,~'= n a n "It ,if=mln~ /(cf. cf W * h l o II), Tl/ a a mi,i-÷,,r¢~ n f twelve ' l - w M . ~ amino oral, Fable mixture of acids (cf. Table II), a mixture mixtm a mixture off +ten B ,vitamins Table llantoin, guanine, hypoxaalthine, xanthine and uric a c i d - - o : i nml of five purine bases (adenine, allantoin, pyrimidines (cytosine, uracil and t h y m i n e - - o . i ml supplying 33 supplying 2o/* each) and three py % alcohol), dicumarol (i mg/ml in 50% alcohol) and adenylic acid aci each), menadione (I mg/ml in 5o% (i mg/ml).
terraNone of these substances was found to take up any oxygen in the presence of terr~ ant mycin under the conditions of the experiment, indicating that the action of the antibiotic was specific for ascorbic acid.
Test/or the presence o/copperr and other metallic impurities in terramycin abo~ In view of the fact that traces of heavy metals, especially copper e can bring about dc acid the antibiotic was tested for the presence of such suc catalytic oxidation of ascorbic wit impurities. This is importantt because terramycin is known to form complexes with erty is actually utilised in the commercial extraction of ( heavy metals and this property itivc brothsL However, the antibiotic failed to give a positi~ the antibiotic from culture brothsL Re[erences p. 509.
igestion with sulphuric acid, whe~ ed to this test. antibiotic was found to be thermo, r fluorescence in the ultraviolet re~ tctions was found to be around 8 plete within two hours. The catai oncentrations as low as I tzg.
ot affect optimal y of the
on o/ascorbic acid by copper and t~ nism of oxidation of ascorbic acid, xidation b y copper was tried on ~ted by terramycin. The results obtained have been summaris~ s ~rcentage inhibition on the endogenous, copper-cataly,sed and t tions of ascorbic acid has been given. Out of the nineteen ni st [roxyquinoline and penicillin G exerted an inhibitory inhibit~ aetio: 7sed b y terramycin. Sodium cyanide, glutathione and cysteine ~ the auto- and copper-catalysed oxidation of ascorbic ascorbk acid. A1 pting dicumarol, menadione and allantoin, had antioxidant an ~sis.
f several c action [I where atalysed ied only xidation nhibited reagents copper
TABLE II EFFECT
OF CERTAIN
SUBSTANCES
ON
OXIDATION
O]F A S C O R B I C O
% in Reagent
I. 2. 3. 45. 6. 7. 8. 9. io. ii. 12. 13. 14 . 15. 16. 17. 18. 19. 20.
Sodium cyanide Bo ric a c i d Glutathione Cysteine-HC1 Y e a s t nucleic acid 8-Hydroxyquinoline M i x t u r e of a m i n o acids M i x t u r e of B v i t a m i n s Y e a s t a d e n y l i c acid Adenine sulphate Guanine dihydrochloride Hypoxanthine Allantoin Uric acid Xanthine M i x t u r e of p y r i m i d i n e s Menadione Dicumarol Penicillin G Urea
Effective Concentration in reaction mixture
o.o 3 M 0.oo 3 M 0.2 m g 0.2 m g I .o m g -3 3' IO M
Endogenous
IOO o ioo 95 4° 16
85 i.o o.i o.i o. I o.i o.I o.I
mg mg mg mg mg mg mg
I mg Img I mg o.I
1"[//
5° ioo ioo ioo 95 3.o 65 94 82 o o 72 o
Copperoxidation
IO0
Terramycin oxidation
O
0
O
IO0
o
I00
o
IO0
8o 7° 33 3° 84 87 82 3.0 80 92 86 o
O
3° o
o o o O o o O o O o
o
O
o
3°
o
o
* A t n i n o a c i d s (Alanine, a s p atrtic r t i c acid, arginine, c y s t i n e , glycine, g l u t a m i c acid, lysine, p~roline, rolin p h e n y l a l a n i n e , v a l i n e t h r e o nnine, i n e , a n d serine 231/~g each). B. v i t a m i n s ( t h i a m i n , riboflavin a v i n , n i a c i n a m i d e , cal. p a n t o t h e n a t e , p y r i d o x a l , inositol, p0-amino-amim benzoic acid, biotin, folic acid id a n d B12, 4 Ng each). ** ~ P y r i m i d i n e s (cytosine, t h y mline i n e a n d u r a c i l 33/~g each). ~#
.
.
Re/erences p. 509. 37
I)IS('VSSION aental evidence it is a p p a r e n t th n h e r e n t p r o p e r t y of the antibiotic i involved in this function is ruled ations, fails to give positive colour rations of copper as low as o.16 ~t ~ne a n d purine bases does not aff~ eas its oxidation b y copper is ce )f the oxidation of ascorbic acid b
ation of )ssibility ;e I. the aich was :lition of n of the :evented a n d the
Vlanifestation of this oxidizing nt in p r o p e r t y b y the aantibiotic e~ tte t h a t the reaction is catalytic in n a t u r e . Since the a n t i b i o t i ic a n t i b a c t e r i a l a n d fluorescent properties after catalysing cata th eem to be chemically involved. A t t e m p t s made to elucidate eh tl :alysis b y the use of inhibitors have n o t been very fruitful. fr N~ excepting penicillin G a n d 8 - h y d r o x y q u i n o l i n e h a d a n y a n t bic acid in presence of the antibiotic. E v e n these ttwo ~ reagen ction to the v i t a m i n . ?he catalytic a c t i v i t y of t e r r a m y c i n reported in the,, ese studies ghly specific for ascorbic acid. If the a n t i b i o t i c were tto functic tion catalyst, f o r m a t i o n of hydrogen peroxide as a seconda~ t be indicated. Since hydrogen peroxide even in low concentra t is conceivable t h a t the catalytic p r o p e r t y of tterrar . .e . . .r . . .r a m y j c . .i n . . . rel . yc . be related to its mode of action.
s would charac, it does chanism lhibitors orion on / partial
. . . . . . . . . .
hown to :idation product ic to the . . . s. t.u. d y is
A C K N O W L E D G E M E N T S
The ,'he authors' grateful t h a n k s are due to Dr. D. L. SHRIVASTAVA, SHR Assistant Direct¢ Director (Biochemistr hemistry) a n d Dr. B. MUKERJI, Director, Central Drug g Research I n s t i t u t e , fc for their keen interest in this work.
SUMMARY I. Six crystalline antibiotics~, penicillin G, dihydrostreptomycin sulphate, aureomycin hydrq [rochloride, chloramphenicol, neomycin ycin sulphate and terramycin hydrochloride were tested for t h e i r oxidation on ascorbic acid. Only terramycin was found to catalyse the reaction. 2. Terramycin has been shown Jcn to be free from traces of metals. The catalytic activity iss tthermq hermostable. The antibiotic activity and fluorescent property of terramycin are not altered by its catalys Tsis of ascorbic acid oxidation. 3. Terramycin oxidizes ascorbic rbic to dehydroascorbic acid without liberating any carbon dioxid lioxide. pH optimum of the reaction is aroum ,round 8. MICHA•LIS' constant of the reaction is O.OLO3M whereas where~ the catalyst substrate dissociation n constant is 5' lO-6. 4. Sodium cyanide, boric acid :id, ghitathione, cysteine, yeast nucleic acid, purines, pyrimidine imidines, amino acids, vitamins of B cornI?lex, menadione and dicumarol do not inhibit the oxidation of :a scorbic acid by terramycin, whereas :eas 8-hydroxyquinoline and penicillin G exert 30% inhibition. 5. Significance of the present ~nt observations on the mode of action of terramycin is briefly briefl discussed. Re]erences p. 509 .
x
. x~.]bux~u J.,.J 2xb ].13 D I
J. L 1~.i~.21J.,4l| t.~1~
R]~SUMI~ n de six a n t i b i o t i q u e s cristallisds (PenJ a u r d o m y c i n e , chloramphdnicol, sulfate ltion de l'acide ascorbique. Seule la l
[ate de diet chlora t a l y s e la
ine ne r e n f e r m a i e n t p a s de traces md vitd a n t i b i o t i q u e et leur fluorescence n l'acide ascorbique. a s c o r b i q u e en acide d d h y d r o a s c o r b i q t de la rdaction est d ' e n v i r o n 8. L a cons tion du c o m p l e x e c a t a l y s e u r - s u b s t r a t e ie borique, le g l u t a t h i o n , la cystdine, ~s aminoacides, les v i t a m i n e s du group, )n de l'acide ascorbique p a r la terrain lroxy-quinoline et la pdnicilline G p r o d u i s e n t u n e inhibition C de 30%. 5. L a s i g n i f i c a t i o n des o b s e r v a t i o n s p r d c d d e n t e s p o u r le moq:le d'actio; ~ment discutde.
tr activitd )difides a u tion d ' a n -IAELIS e s t
q u e de la lione et le ntraire, la nycine e s t
ZUSAMMENFASSUNG I. Die W i r k u n g y o n 6 kristaUisierten A n t i b i o t i k a , Penicilli Penicillin G, D i h 3 rcinsulfat, )mycinchlorhydrat, Chloramphenicol, Neomycinsulfat and Terramyc : bei d e r a t i o n y o n Ascorbins~ture w u r d e geprfift. E s w u r d e g e f u n d e n , d a s s n u t T, Reaktion vsiert. 2. E s w u r d e gezeigt, d a s s T e r r a m y c i n frei y o n M e t a l l s p u r e n :ist. Die ka t i v i t ~ t ist tostabil. Die a n t i b i o t i s c h e Aktiviti~t u n d die Fluoreszenzeigce n s c h a f t d~ is w e r d e n ;r K a t a l y s e d e r A s c o r b i n s i i u r e o x y d a t i o n n i c h t veri~ndert. 3. T e r r a m y c i n o x y d i e r t Ascorbins~kure zu Dehydroaseorbins~,trare o h n e F. a Kohlend. D a s p H - O p t i m u m der R e a k t i o n liegt bei 8. Die MICHAEL]. I s k o n s t a n t , n betriigt 3 M , die K a t a l y s a t o r - S u b s t r a t - D i s s o z z i a t i o n s k o n s t a n t e 5' 1i o0 - - 6 t- N a t r i u m c y a n i d , BorsAure, G l u t a t h i o n , Cystein, Hefenucleinsiiure, P u r i n e , P yrrimidine, rimidir os~.uren, V i t a m i n e des B - K o m p l e x e s M e n a d i o n u n d Dicum~carol h e m m e n die O x y d a t i o n vv o n A s c o r bbinsiiure ins m i t T e r r a m y c i n nicht, d a g e g e n fiben 8-Oxyquinolin uinolin a n d Penicillin G eine H e m m uu n g v a n 3 0 % aus. 5. Die B e d e u t u n g d e r v o r l i e g e n d e n B e o b a c h t u n g e n a u f die A 5. A r t der W i r k u n g des T e r r a m y c"clns i wird k u r z b e s p r o c h e n .
REFERENCES 1 K . L . ,. A R O R A AND C. R . K R I S H N A M U R T I , u n p u b l i s h e d w o r k . Z J . R . • P O R T E R , Bacterial Chemistry and Physiology, J o h n W i l e y & Sons (1947) 751 . 3 J . H . • ROE AND M. J. OESTERLING, J. Biol• Chem., 152 (1944) 511 II. 4 F. J. WELCHER, Organic Analytical ~ical Reagents, C. V a n N o s t r a n d Co., N e w Y o r k 1948, Vol. I I I p.~-519. 51 5 A. T. D U D A N I AND S. C. A G A R WCALA, AL J. Sci. Indian Res., l i B (1952) 551. 6 K . V • GIRI AND P. SESHAGIRI RA [{AO, Proc. Indian Acad. Sci. B. 24 (1947) 264. 7 p. REGNA PETER, R. PASTERNA ACK, I . A . SOLOMONS, K O T A R O M U R A I , R . L . W A G N E R , A . B AVEL BAVELY, F• A. H O C H S T E I N , P. N. GORDE~ EN, G . H E S S , A . E . T I M R E C K , K . J. B R U N N I N G S AND W . A . L A Z l Et.R, Bull. World• Health Org. 6 (1952) 123. Received
October
3 o t h , I9~ 953
ACID
BY TERRA1
by ANI
ANn C. R. K R I S H N A M U R T I
lral Drug Research Institute, Lucknow, (India)
While s t u d y i n g t h e effect of a n t i b i o t i c s on t h e c y~tochrome tocl ox m s u a l l y h i g h e n d o g e n o u s o x y g e n u p t a k e w a s o b s e r v e d in con ne C, t e r r a m y c i n a n d a s c o r b i c a c i d w e r e i n c u b a t e d tt,o g e t h e r i~ iae. T h i s e n h a n c e d o x y g e n c o n s u m p t i o n w a s subseqt u e n t l y e, e o x i d a t i o n of a s c o r b i c a c i d b y t e r r a m y c i n t o t h e d e h y d r o fi r t a n c e of a s c o r b i c a c i d in t h e m a i n t e n a n c e of ooxid~ xidation red gical s y s t e m s ~, it w a s c o n s i d e r e d of i n t e r e s t to s t u d y in s o ~ r e d in t h i s i n t e r e s t i n g c a t a l y t i c p r o p e r t y of t h e aanti~ ntibiotic.
,,f h e a r t 1, [ch c y t o :e of t h e be due w of t h e n t i a l s of factors
EXPERIMENTAL
Antibiotics and other chemicals
['he following crystalline antibiotic obtained through the courtesy co The of their respective m man anufacturers ers were used in this study. Penicillin G (Indian Penicillin Committee), C Dihydrostreptomyc rcin zte (Glaxo Laboratories), Aureomycin hydrochloride (Lederl sulphate Lederle Laboratories), Chloramphenic ~henicol (Parkee Davis & Company), Neomycin sulphate (Upjohn & Compan Lpany) and Terramycin hydrochlori~ rochloride (Charles Pfizer & Company). Ascorbic ~scorbic acid and other chemicals employed were of reage~ gent grade purchased from Britifish Drug Houses Ltd., Charles Pfizer or Nutritional Bio-chemicals. Manometry
Conventional Warburg techniques es were adopted using flasks with centre cups and single side arn= ~rms. Analytical method
Ascorbic acid was estimatedfl according to ROE AND OESTERLING3. Spot tests for copper in the dru ]rugs used were carried out with dithiozonc reagent 4 (io mg in too ml T chloroform). Antibiotic assay
Terramycin was tested by the :he standard cup assay method ~ with S. aureus (Oxford) and E. ec cell as test organisms. Fluorimetry
The fluorescence of terramy 'cin was measured in a Lumetron Fluorimetcr 4o2 E using the tilt filter accessories for riboflavin. * An abstract of this paper was as :presented at a symposium on "Antibacterial substances from soil, so plants and other sources" held under the auspices of the Pharmaceuticals and Drugs Committe9e~ Council of Scientific and Industrial ial Research, at Bombay on the 26th March I953. Re/erenees p. 509.
RESULTS
(m ascorbic acid d to pH 7 were tipped in from the phate buffer pH 7.o and I ml ot were taken over a period of one he various reacting systems in 6o
the flask solution ) mimlte given ill
TABLE I F ASCORBIC ACID IN P R E S E N C E
A ntibiotic added
(Control) Aureomycin hydrochloride Chloramphenicol Neomycin sulphate Dihydrostreptomycin sulphatc Penicillin G Terramycin hydrochloride Water
O x. y. g .~ -. c .~ d. . microliters
25.0 24.2 25.7 21. 5 30.0 18.6 168.4
OF 6 ANt
- - ~ o / o g . , , ~ , , .... or inhibition
v+. -+
3.: 2.~ i4., 2o.i 25.q 573 -*
ill the antibiotics were found to affect the oxidation one ( way ( . Aureol, neomycin and penicilhn inhibited the oxidation, the maximui g shown nicillin G. Dihydrostreptomycin and terramycin accelerated acce tile rate ot OXldatloi tt :idation. atalytic action of terramycin was most pronounced, subsequent work was, therether( fore, confined to this antibiotic.
qcily o/the reaction with respect to substrate Specificity simi action on other reducin g Inn order to find out whether terramycin exerted a similar subst~ were tested: agentss and chemicals of biological interest following substances ~lucose, gluconic acid, lactic acid, pyruvic acid, sorbitol, sorbose, quercitol, glutathion tathione, Glucose le hydrochloride, sodium bisulphite, sodium thiosulphate, phenylenediamine, p sodium nitrite, nitrit cysteine din, cholesterol and vegetable lecithin (all these substances in I mg/ml adjusted to pH 7.0 adrenalin 7.0) ; -,,~'= n a n "It ,if=mln~ /(cf. cf W * h l o II), Tl/ a a mi,i-÷,,r¢~ n f twelve ' l - w M . ~ amino oral, Fable mixture of acids (cf. Table II), a mixture mixtm a mixture off +ten B ,vitamins Table llantoin, guanine, hypoxaalthine, xanthine and uric a c i d - - o : i nml of five purine bases (adenine, allantoin, pyrimidines (cytosine, uracil and t h y m i n e - - o . i ml supplying 33 supplying 2o/* each) and three py % alcohol), dicumarol (i mg/ml in 50% alcohol) and adenylic acid aci each), menadione (I mg/ml in 5o% (i mg/ml).
terraNone of these substances was found to take up any oxygen in the presence of terr~ ant mycin under the conditions of the experiment, indicating that the action of the antibiotic was specific for ascorbic acid.
Test/or the presence o/copperr and other metallic impurities in terramycin abo~ In view of the fact that traces of heavy metals, especially copper e can bring about dc acid the antibiotic was tested for the presence of such suc catalytic oxidation of ascorbic wit impurities. This is importantt because terramycin is known to form complexes with erty is actually utilised in the commercial extraction of ( heavy metals and this property itivc brothsL However, the antibiotic failed to give a positi~ the antibiotic from culture brothsL Re[erences p. 509.
igestion with sulphuric acid, whe~ ed to this test. antibiotic was found to be thermo, r fluorescence in the ultraviolet re~ tctions was found to be around 8 plete within two hours. The catai oncentrations as low as I tzg.
ot affect optimal y of the
on o/ascorbic acid by copper and t~ nism of oxidation of ascorbic acid, xidation b y copper was tried on ~ted by terramycin. The results obtained have been summaris~ s ~rcentage inhibition on the endogenous, copper-cataly,sed and t tions of ascorbic acid has been given. Out of the nineteen ni st [roxyquinoline and penicillin G exerted an inhibitory inhibit~ aetio: 7sed b y terramycin. Sodium cyanide, glutathione and cysteine ~ the auto- and copper-catalysed oxidation of ascorbic ascorbk acid. A1 pting dicumarol, menadione and allantoin, had antioxidant an ~sis.
f several c action [I where atalysed ied only xidation nhibited reagents copper
TABLE II EFFECT
OF CERTAIN
SUBSTANCES
ON
OXIDATION
O]F A S C O R B I C O
% in Reagent
I. 2. 3. 45. 6. 7. 8. 9. io. ii. 12. 13. 14 . 15. 16. 17. 18. 19. 20.
Sodium cyanide Bo ric a c i d Glutathione Cysteine-HC1 Y e a s t nucleic acid 8-Hydroxyquinoline M i x t u r e of a m i n o acids M i x t u r e of B v i t a m i n s Y e a s t a d e n y l i c acid Adenine sulphate Guanine dihydrochloride Hypoxanthine Allantoin Uric acid Xanthine M i x t u r e of p y r i m i d i n e s Menadione Dicumarol Penicillin G Urea
Effective Concentration in reaction mixture
o.o 3 M 0.oo 3 M 0.2 m g 0.2 m g I .o m g -3 3' IO M
Endogenous
IOO o ioo 95 4° 16
85 i.o o.i o.i o. I o.i o.I o.I
mg mg mg mg mg mg mg
I mg Img I mg o.I
1"[//
5° ioo ioo ioo 95 3.o 65 94 82 o o 72 o
Copperoxidation
IO0
Terramycin oxidation
O
0
O
IO0
o
I00
o
IO0
8o 7° 33 3° 84 87 82 3.0 80 92 86 o
O
3° o
o o o O o o O o O o
o
O
o
3°
o
o
* A t n i n o a c i d s (Alanine, a s p atrtic r t i c acid, arginine, c y s t i n e , glycine, g l u t a m i c acid, lysine, p~roline, rolin p h e n y l a l a n i n e , v a l i n e t h r e o nnine, i n e , a n d serine 231/~g each). B. v i t a m i n s ( t h i a m i n , riboflavin a v i n , n i a c i n a m i d e , cal. p a n t o t h e n a t e , p y r i d o x a l , inositol, p0-amino-amim benzoic acid, biotin, folic acid id a n d B12, 4 Ng each). ** ~ P y r i m i d i n e s (cytosine, t h y mline i n e a n d u r a c i l 33/~g each). ~#
.
.
Re/erences p. 509. 37
I)IS('VSSION aental evidence it is a p p a r e n t th n h e r e n t p r o p e r t y of the antibiotic i involved in this function is ruled ations, fails to give positive colour rations of copper as low as o.16 ~t ~ne a n d purine bases does not aff~ eas its oxidation b y copper is ce )f the oxidation of ascorbic acid b
ation of )ssibility ;e I. the aich was :lition of n of the :evented a n d the
Vlanifestation of this oxidizing nt in p r o p e r t y b y the aantibiotic e~ tte t h a t the reaction is catalytic in n a t u r e . Since the a n t i b i o t i ic a n t i b a c t e r i a l a n d fluorescent properties after catalysing cata th eem to be chemically involved. A t t e m p t s made to elucidate eh tl :alysis b y the use of inhibitors have n o t been very fruitful. fr N~ excepting penicillin G a n d 8 - h y d r o x y q u i n o l i n e h a d a n y a n t bic acid in presence of the antibiotic. E v e n these ttwo ~ reagen ction to the v i t a m i n . ?he catalytic a c t i v i t y of t e r r a m y c i n reported in the,, ese studies ghly specific for ascorbic acid. If the a n t i b i o t i c were tto functic tion catalyst, f o r m a t i o n of hydrogen peroxide as a seconda~ t be indicated. Since hydrogen peroxide even in low concentra t is conceivable t h a t the catalytic p r o p e r t y of tterrar . .e . . .r . . .r a m y j c . .i n . . . rel . yc . be related to its mode of action.
s would charac, it does chanism lhibitors orion on / partial
. . . . . . . . . .
hown to :idation product ic to the . . . s. t.u. d y is
A C K N O W L E D G E M E N T S
The ,'he authors' grateful t h a n k s are due to Dr. D. L. SHRIVASTAVA, SHR Assistant Direct¢ Director (Biochemistr hemistry) a n d Dr. B. MUKERJI, Director, Central Drug g Research I n s t i t u t e , fc for their keen interest in this work.
SUMMARY I. Six crystalline antibiotics~, penicillin G, dihydrostreptomycin sulphate, aureomycin hydrq [rochloride, chloramphenicol, neomycin ycin sulphate and terramycin hydrochloride were tested for t h e i r oxidation on ascorbic acid. Only terramycin was found to catalyse the reaction. 2. Terramycin has been shown Jcn to be free from traces of metals. The catalytic activity iss tthermq hermostable. The antibiotic activity and fluorescent property of terramycin are not altered by its catalys Tsis of ascorbic acid oxidation. 3. Terramycin oxidizes ascorbic rbic to dehydroascorbic acid without liberating any carbon dioxid lioxide. pH optimum of the reaction is aroum ,round 8. MICHA•LIS' constant of the reaction is O.OLO3M whereas where~ the catalyst substrate dissociation n constant is 5' lO-6. 4. Sodium cyanide, boric acid :id, ghitathione, cysteine, yeast nucleic acid, purines, pyrimidine imidines, amino acids, vitamins of B cornI?lex, menadione and dicumarol do not inhibit the oxidation of :a scorbic acid by terramycin, whereas :eas 8-hydroxyquinoline and penicillin G exert 30% inhibition. 5. Significance of the present ~nt observations on the mode of action of terramycin is briefly briefl discussed. Re]erences p. 509 .
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R]~SUMI~ n de six a n t i b i o t i q u e s cristallisds (PenJ a u r d o m y c i n e , chloramphdnicol, sulfate ltion de l'acide ascorbique. Seule la l
[ate de diet chlora t a l y s e la
ine ne r e n f e r m a i e n t p a s de traces md vitd a n t i b i o t i q u e et leur fluorescence n l'acide ascorbique. a s c o r b i q u e en acide d d h y d r o a s c o r b i q t de la rdaction est d ' e n v i r o n 8. L a cons tion du c o m p l e x e c a t a l y s e u r - s u b s t r a t e ie borique, le g l u t a t h i o n , la cystdine, ~s aminoacides, les v i t a m i n e s du group, )n de l'acide ascorbique p a r la terrain lroxy-quinoline et la pdnicilline G p r o d u i s e n t u n e inhibition C de 30%. 5. L a s i g n i f i c a t i o n des o b s e r v a t i o n s p r d c d d e n t e s p o u r le moq:le d'actio; ~ment discutde.
tr activitd )difides a u tion d ' a n -IAELIS e s t
q u e de la lione et le ntraire, la nycine e s t
ZUSAMMENFASSUNG I. Die W i r k u n g y o n 6 kristaUisierten A n t i b i o t i k a , Penicilli Penicillin G, D i h 3 rcinsulfat, )mycinchlorhydrat, Chloramphenicol, Neomycinsulfat and Terramyc : bei d e r a t i o n y o n Ascorbins~ture w u r d e geprfift. E s w u r d e g e f u n d e n , d a s s n u t T, Reaktion vsiert. 2. E s w u r d e gezeigt, d a s s T e r r a m y c i n frei y o n M e t a l l s p u r e n :ist. Die ka t i v i t ~ t ist tostabil. Die a n t i b i o t i s c h e Aktiviti~t u n d die Fluoreszenzeigce n s c h a f t d~ is w e r d e n ;r K a t a l y s e d e r A s c o r b i n s i i u r e o x y d a t i o n n i c h t veri~ndert. 3. T e r r a m y c i n o x y d i e r t Ascorbins~kure zu Dehydroaseorbins~,trare o h n e F. a Kohlend. D a s p H - O p t i m u m der R e a k t i o n liegt bei 8. Die MICHAEL]. I s k o n s t a n t , n betriigt 3 M , die K a t a l y s a t o r - S u b s t r a t - D i s s o z z i a t i o n s k o n s t a n t e 5' 1i o0 - - 6 t- N a t r i u m c y a n i d , BorsAure, G l u t a t h i o n , Cystein, Hefenucleinsiiure, P u r i n e , P yrrimidine, rimidir os~.uren, V i t a m i n e des B - K o m p l e x e s M e n a d i o n u n d Dicum~carol h e m m e n die O x y d a t i o n vv o n A s c o r bbinsiiure ins m i t T e r r a m y c i n nicht, d a g e g e n fiben 8-Oxyquinolin uinolin a n d Penicillin G eine H e m m uu n g v a n 3 0 % aus. 5. Die B e d e u t u n g d e r v o r l i e g e n d e n B e o b a c h t u n g e n a u f die A 5. A r t der W i r k u n g des T e r r a m y c"clns i wird k u r z b e s p r o c h e n .
REFERENCES 1 K . L . ,. A R O R A AND C. R . K R I S H N A M U R T I , u n p u b l i s h e d w o r k . Z J . R . • P O R T E R , Bacterial Chemistry and Physiology, J o h n W i l e y & Sons (1947) 751 . 3 J . H . • ROE AND M. J. OESTERLING, J. Biol• Chem., 152 (1944) 511 II. 4 F. J. WELCHER, Organic Analytical ~ical Reagents, C. V a n N o s t r a n d Co., N e w Y o r k 1948, Vol. I I I p.~-519. 51 5 A. T. D U D A N I AND S. C. A G A R WCALA, AL J. Sci. Indian Res., l i B (1952) 551. 6 K . V • GIRI AND P. SESHAGIRI RA [{AO, Proc. Indian Acad. Sci. B. 24 (1947) 264. 7 p. REGNA PETER, R. PASTERNA ACK, I . A . SOLOMONS, K O T A R O M U R A I , R . L . W A G N E R , A . B AVEL BAVELY, F• A. H O C H S T E I N , P. N. GORDE~ EN, G . H E S S , A . E . T I M R E C K , K . J. B R U N N I N G S AND W . A . L A Z l Et.R, Bull. World• Health Org. 6 (1952) 123. Received
October
3 o t h , I9~ 953