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A study of some enzyme systems of PSEUDOMONAS AERUGINOSA Warburton, Roger Hartley 1951

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A STUDY OF SOME ENZYME SYSTEMS OF PSEUDOMONAS AERUGINOSA b y ROGER HARTLEY WARBURTON A THESIS SUBMITTED I N P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE I N AGRICULTURE i n t h e D e p a r t m e n t o f . D a i r y i n g We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e S t a n d a r d r e q u i r e d f r o m c a n d i d a t e s f o r t h e d e g r e e o f MASTER OF SCIENCE I N AGRICULTURE Members p f t h e D e p a r t m e n t o f THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , 1951. ABSTRACT P y r u v a t e h a s b e e n d e t e r m i n e d a t 1 6 , 28 a n d 4.0 h o u r s i n a c u l t u r e o f Pseudomonas a e r u g i n o s a when grown i n a g l u c o s e medium. S i n c e t h r o u g h o u t t h i s e n t i r e p e r i o d t h e o r g a n i s m p o s s e s s e d t h e enzyme s y s t e m c a p a b l e o f r a p i d l y o x i d i z i n g p y r u v a t e , i t was c o n c l u d e d t h a t p y r u v a t e was b e i n g f o r m e d a n d d i s s i m i l a t e d c o n t i n u o u s l y a n d was t h e r e f o r e a n i n t e r m e d i a t e i n t h e o x i d a t i o n o f g l u c o s e . I t was f o u n d t h a t g l u c o s e o x i d a t i o n was n o t i n h i b i t e d by 0 . 0 2 M s o d i u m f l u o r i d e and t h a t p y r u v a t e f o r m a t i o n and u t i l i z a t i o n c o n t i n u e d u n i m p a i r e d i n t h e p r e s e n c e o f t h e i n h i b i t o r . T h i s w o u l d i n d i c a t e t h a t e n o l a s e i s n o t c o n c e r n e d i n t h e f o r m a t i o n o f p y r u v a t e b y P . a e r u g i n o s a a n d t h e r e f o r e t h e E m b d e n - M e y e r h o f scheme o f g l u c o s e d e g r a d a t i o n does n o t f u n c t i o n i n t h i s o r g a n i s m . To s t u d y t h e i n i t i a l r e a c t i o n i n t h e d e g r a d a t i o n o f g l u c o s e by g l u c o s e o x i d a s e o f P . a e r u g i n o s a a p r e p a r a t i o n o f d r i e d c e l l s was e m p l o y e d u s i n g a 24 h o u r o l d c u l t u r e a n d d r y i n g them o v e r p h o s p h o r u s p e n t o x i d e . T h e d r i e d c e l l s a l t h o u g h c a p a b l e o f o x i d i z i n g g l u c o s e t o 2 k e t o g l u c o n i c a t p H 7 . 2 , were u n a b l e t o o x i d i z e g l u c o s e f u r t h e r t h a n g l u c o n i c a c i d a t pH7 . 5 . I t was f o u n d t h a t t h e enzyme was n o t i n h i b i t e d b y m a l o n a t e , i o d o a c e t a t e o r a r s e n a t e b u t t h a t t h e s y s t e m was i m p a i r e d by c y a n i d e and s o d i u m a z i d e . The i n h i b i t i o n b y c y a n i d e w o u l d i n d i c a t e t h a t c y t o c h r o m e o x i d a s e o r a s e c o n d c y a n i d e s e n s i t i v e c a r r i e r i s i n v o l v e d . The a c t i o n o f p r o t e o l y t i c e n z y m e s , l i g h t and t e m p e r a t u r e were d e t e r m i n e d . The enzyme t r y p s i n was f o u n d t o d e s t r o y t h e g l u c o s e o x i d a s e a c t i v i t y w h i l e t h e e f f e c t o f p e p s i n a n d p a p a i n c o u l d n o t be e s t a b l i s h e d . - 2 -Strong light was found to interfere with the f u l l enzyme activity and a temperature of 55°C« completely destroyed the enzyme. Temperatures of 37°C. and 45°C. after 60 minutes reduced the oxygen uptake for the enzyme by as much as 50%. The separation of the enzyme into two fractions, co-enzyme and apo-enzyme, was effected by ammonium sulphate precipitation and dialysis against distilled water. It was found that these two fractions alone, or when combined, could not mediate the reaction.glucose to gluconic acid. The co-enzyme was therefore concluded to be dialysable. Addition of known compounds to the fraction showed magnesium and manganese as ions capable of restoring the activity of the enzyme. Magnesium was found to be the more active of the two substances in restoring the enzymes activity. This would indicate that magnesium was the co-cnzyme and was capable of being replaced by manganese. ACKNOWLEDGEMENT I w i s h t o e x p r e s s ray s i n c e r e t h a n k s t o D r . J . J . R . C a m p b e l l f o r h i s e n c o u r a g e m e n t a n d a s s i s t a n c e d u r i n g t h e c o u r s e o f t h i s w o r k , a n d t o Dean B . A . E a g l e s f o r h i s v a l u a b l e s u g g e s t i o n s a n d a s s i s t a n c e t h r o u g h o u t t h i s s t u d y . 1 a l s o w i s h t o t h a n k t h e N a t i o n a l R e s e a r c h C o u n c i l f o r a g r a n t t o c a r r y o u t p a r t o f t h i s w o r k . R . H . W . TABLE OF CONTENTS PART I THE IDENTIFICATION OF PYRUVATE AS AN INTERMEDIATE IN GLUCOSE OXIDATION Introduction 1 Methods 3 Bacteriological 3 Chemical . . . . . . 4 Experimental 5 Table 1 6 Figure 1 7 Figure 2 8 Discussion ...10 Summary 11 ..PART.II THE ISOLATION OF GLUCOSE OXIDASE AND A STUDY OF ITS PROPERTIES Introduction 12 Methods .... . . . . . 15 Bacteriological 15 Chemical 16 Experimental Anaerobic studies 17 Table 2 18 Table 3 19 Table 4 22 Aerobic experiments 23 Figure 3 24 Figure 4 25 Table 5 26 Enzyme action . . . . . 27 Effect of light 28 Figure 5 29 Effect of temperature 30 Action of inhibitors 30 Table 6 32 Table 7 33 Coenzyme 34 Procedure for separating glucose oxidase into two fractions 35 Figure 6 . . . 36 Discussion ..38 Summary BIBLIOGRAPHY PART I . THE I D E N T I F I C A T I O N OF PYRUVATE AS AN INTERMEDIATE I N GLUCOSE OXIDATION INTRODUCTION The m e t a b o l i s m o f l a c t i c a c i d f o r m i n g o r g a n i s m s ' o r t i s s u e s h a s b e e n s t u d i e d i n g r e a t d e t a i l a n d t h e pa thway o f g l u c o s e f e r m e n t a t i o n i s known t o go t h r o u g h a s e r i e s o f p h o s p h o r y l a t e d i n t e r m e d i a t e s a n d p y r u v i c a c i d . T h i s scheme o f m e t a b o l i s m i s known a s t h e E m b d e n - M e y e r h o f . T h i s p a t h w a y o p e r a t e s a n a e r o b i c a l l y a n d , t h e r e f o r e , c o n s i d e r a b l e d i s c u s s i o n and e x p e r i m e n t a l w o r k h a s b e e n done t o d e t e r m i n e w h e t h e r o r n o t t h i s scheme a l s o o p e r a t e s i n a e r o b i c o r g a n i s m s . I n r e c e n t y e a r s i t h a s b e e n g e n e r a l l y a c c e p t e d t h a t a e r o b i c t i s s u e a n d m i c r o o r g a n i s m s d i s s i m i l a t e g l u c o s e b y way o f t h e E m b d e n - M e y e r h o f (22) a s f a r a s p y r u v a t e and f r o m t h i s p o i n t o n t h e p y r u v a t e i s o x i d i z e d b y way o f t h e K r e b s t r i c a r b o x y l i c a c i d c y c l e ( 1 6 ) . M o r e r e c e n t w o r k b y L i p m a n n (18) d e m o n s t r a t e d t h a t p y r u v a t e i s f i r s t o x i d i z e d t o a c e t y l p h o s p h a t e and t h e n a c e t y l p h o s p h a t e e n t e r s t h e K r e b s c y c l e . D i c k e n s (7 ) a n d L i p m a n n (17) s u g g e s t e d a pa thway t h a t d i f f e r e d f r o m t h e E m b d e n - M e y e r h o f f e r m e n t a t i o n b r e a k d o w n , p o s t u l a t i n g g l u c o s e-6 - p h o s p h a t e a n d p h o s p h o g l u c o n i c a c i d a s t h e f i r s t i n t e r m e d i a t e s i n t h e scheme. L o c k -w o o d , T a b e n k i h a n d Ward (20) i s o l a t e d g l u c o n i c and 2 k e t o g l u c o n i c a c i d s f r o m s p e c i e s o f Pseudomonas a n d P h y t o m o n a s . L a t e r L o c k w o o d a n d S t o d o l a (19) i s o l a t e d * * . k e t o g l u t a r a t e f r o m . s u b m e r g e d f e r m e n t a t i o n c u l t u r e s o f P . f l u o r e s c e n s when g r o w n o n g l u c o s e , g l u c o n i c a c i d o r 2 k e t o g l u c o n i c a c i d . N e y (25) d e m o n s t r a t e d t h e p r e s e n c e o f 2 k e t o g l u c o n a t e i n t h e s u p e r n a t a n t o f P . a e r u g i n o s a grown o n g l u c o s e medium u n d e r p h y s i o l o g i c a l l y a b n o r m a l c o n d i t i o n s . R e c e n t w o r k by N o r r i s and C a m p b e l l (26) h a s d e m o n s t r a t e d t h a t P . a e r u g i n o s a o x i d i z e s g l u c o s e b y a p a t h w a y w h i c h d i f f e r s f r o m a n y p r e v i o u s l y r e p o r t e d f o r t i s s u e o r m i c r o o r g a n i s m s . They e s t a b l i s h e d t h a t glucose i s oxidized to gluconic acid which i n turn i s oxidized to 2 keto-gluconic acid (26), (32). They also showed acetate to be an intermediate (2). The major importance of this pathway to this organism was confirmed when they found that none of the members of the Embden-Meyerhof scheme could be detected. In order to establish whether or not pyruvate i s an intermediate in the breakdown of glucose by P. aeruginosa the.present study was undertaken. - 3 -METHODS B a c t e r i o l o g i c a l Pseudomonas a e r u g i n o s a 9027 was u s e d a s t h e t e s t o r g a n i s m . S t o c k c u l t u r e s were m a i n t a i n e d o n l i v e r e x t r a c t g e l a t i n c o n s i s t i n g o f 10$ t r y p t o n e , 0.3% K2HPO4, 0.1% g l u c o s e , 0.3% g l y c e r o l , 10% l i v e r e x t r a c t , 0.5% a g a r a n d 2.0% g e l a t i n . The medium was a d j u s t e d t o pH7.2 a n d d i s p e n s e d I n t e s t t u b e s p r i o r t o s t e r i l i z a t i o n . G r o w t h was i n i t i a t e d o n t h e l i v e r . e x t r a c t g e l a t i n a g a r a t 30 ° C . p r i o r t o s t o r a g e u n d e r r e f r i g e r a t i o n c o n d i t i o n s . F o r e x p e r i m e n t a l u s e , t h e c u l t u r e was c a r r i e d t h r o u g h t h r e e t r a n s f e r s a t 24. h o u r p e r i o d s i n S u l l i v a n ' s medium (34-) and t h e n i n o c u l a t e d i n t o g l u c o s e m i n e r a l medium, t h e medium u s e d f o r g r o w i n g c e l l s f o r W a r b u r g e x p e r i m e n t s . The c o m p o s i t i o n o f t h e m i n e r a l medium was 0.3% N H ^ H ^ P O ^ , 0.1% MgSO^ .7HgO,. 0.5% c a r b o h y d r a t e a n d 0.5 ppm. o f i r o n a s f e r r o u s s u l p h a t e . The g l u c o s e m i n e r a l medium was d i s p e n s e d i n 200 m l . q u a n t i t i e s i n R o u x f l a s k s . A 10% s o l u t i o n o f K ^ P O ^ was a d d e d t o a f i n a l c o n c e n t r a -t i o n o f 0.3% a f t e r s t e r i l i z a t i o n . The W a r b u r g a p p a r a t u s was e m p l o y e d f o r t h e s t u d i e s i n t e r m e d i a t e m e t a b o l i s m . C e l l s were h a r v e s t e d a t p e r i o d s o f 16, 28 and 40 h o u r s and made t o v o l u m e b y a d d i n g d i s t i l l e d w a t e r . A 0.2 c c . s ample o f t h i s s u s p e n s i o n when d i l u t e d t o 10 c c . r e c o r d e d 74% l i g h t t r a n s m i s s i o n when p l a c e d i n t h e F i s c h e r E l e c t r o p h o t o m e t e r . The same c o n c e n t r a t i o n o f c e l l s was u s e d i n a l l W a r b u r g c u p s . W a r b u r g c u p s c o n t a i n e d 1.5 m l . o f S o r e n s o n ' s p h o s p h a t e b u f f e r pH7.5 and 0.5 m l . c e l l s i n t h e m a i n c o m p a r t m e n t . Sul>-s t r a t e s t o be t e s t e d were t i p p e d i n ^from t h e s i d e a r m . The c e n t e r w e l l c o n t a i n e d 0.15 m l . o f 20% K0H f o r t h e a b s o r p t i o n o f c a r b o n d i o x i d e . The t o t a l v o l u m e o f f l u i d i n t h e c u p s was 3.15 m l . Warburg substrates were added to a concentration of 0.5 ml. equivalent to 18 uM. of oxygen. - A -P y r u v a t e d e t e r m i n a t i o n s w e r e c a r r i e d o u t o n t h e g r o w t h medium a f t e r t h e c e l l s h a d b e e n removed b y c e n t r i f u g i n g . A f t e r a d j u s t m e n t t o p H 7 . 2 , t h e medium was c o n c e n t r a t e d i n v a c u o a t 3 9 ° C . t o one t e n t h o f i t s o r i g i n a l v o l u m e . I n o r d e r t o e n s u r e a t l e a s t a s g r e a t a c o n c e n t r a t i o n o f p r o t e i n i n t h e u n i n o c u l a t e d c o n t r o l f l a s k s a s i n t h e e x p e r i m e n t a l f l a s k s , c e l l s h a r v e s t e d f r o m 20 m i s . o f g r o w t h medium w e r e added t o t h e c o n t r o l f l a s k . C h e m i c a l The t e c h n i q u e o f p a p e r c h r o m a t o g r a p h y was e m p l o y e d f o r t h e d e t e c t i o n o f p y r u v a t e u s i n g t h e 2-A d i n i t r o p h e n y l h y d r a z o n e p r o c e d u r e o f C a v a l l a n i , F r a n t a l i , a n d T o s c h i ( 4 ) , Whatman #1 f i l t e r p a p e r was used and t h e a s c e n d i n g c h r o m a t o g r a p h s were i r r i g a t e d f o r a p e r i o d o f 17 h o u r s a t room t e m p e r a t u r e . C o n f i r m a t i o n o f t h e p r e s e n c e o f p y r u v a t e i n t h e c o n c e n t r a t e was o b t a i n e d b y P r o c e d u r e B o f F r i e d e m a n n and Haugen ( l l ) u s i n g t o l u e n e a s t h e s o l v e n t . F i l t e r #525 was u s e d f o r t h e e l e c t r o p h o t o m e t e r r e a d i n g s . A l l s o l v e n t s u s e d were r e a g e n t g r a d e , a n d p y r u v i c a c i d was p r e p a r e d b y t h e method o f R o b e r t s o n ( 3 1 ) • - 5 -EXPERIMENTAL Analysis of the glucose medium after P. aeruginosa had been growing for 16, 28 and 40 hours revealed that pyruvate was present over the entire 24 hour interval (Table l ) . The 2-4 dinitrophenylhydrazone of pyruvic acid moved as two distinct spots in the acidic solvents. By their melting point and by paper chromatography, the lower Rf value was found to be the free hydrazone of pyruvic acid and the higher Rf value the ethyl ester of pyruvic acid. This characteristic served to simplify the identification of pyruvate. It was found that cells harvested at 16, 28 and 40 hours were capable of oxidizing pyruvate immediately, when tested in the Warburg respirometer (Figure l ) . Since pyruvic acid is detectable in the glucose growth medium for a 24 hour period (16-40 hours) and since the organism has the enzyme system necessary for the oxidation of this acid as shown in Figure 1, i t is to be concluded that throughout the 24 hour interval, pyruvate is being formed and oxidized continuously and is therefore, regarded as an intermediate in the oxidation of glucose. The demonstration that pyruvic acid is an intermediate in the oxidation of glucose by P. aeruginosa might be interpreted as being evidence for an Embden-Meyerhof system. However, in agreement with Barron and Friedemann (l) i t was found that the oxidation of glucose by this organism was not inhibited by 0.021 sodium fluoride. It was also found that when the organism was grown in glucose medium to which fluoride had been added to a concentration of 0.02M it was again possible to detect pyruvate at 16, 28 and 40 hours. Cells harvested from these fluoride media were found to have an unimpaired ability to oxidize glucose .or pyruvate. - 6 -TABLE 1. Rf Values of the 2 -4 Di N 0 2 Phenyl Hydrazones Formed from the Growth Media. 1 2 3 •43 .14 2-4 di N02 phenyl Oxaloacetate •47 .47 hydrazones of °< Ketoglutarate • 51 .11 .16 reference .45 .46 substances Pyruvate .61 .65 .62 .46 .47 2-4 di N 0 2 phenyl at 16 hours .62 .66 .63 hydrazones formed •45 .47 with glucose at 28 hours .61 .65 .62 growth medium .46 .48 at 40 hours . 61 .66 .63 .46 .46 2-4 di NO, phenyl at 16 hours .6? .66 .62 hydrazohes formed •4.6 •48 with glucose growth at 28 hours .61 .66 .63 medium containing .46 .48 fluoride at 40 hours .62 .66 . 63 Solvent 1 . n butyl alcohol saturated with 3% acetic acid. 2 . n butyl alcohol saturated with 3% ammonia. 3. n butyl alcohol 50% ethyl alcohol 10% distilled water 40%. NB Two Rf values are recorded for oxalacetate indicating partial break-down to pyruvic acid. - 7 -3 0 6 0 9 0 MINUTES Fig. 1. Pyruvate oxidation by cells grown on glucose mineral medium. Curves A, B and C^pyruvate oxidation, by 16 hour cells, 28 hour cells and 40 hour cells respectively. - 3 -/ HOURS ' F i g . 2. The accumulation of pyruvic a c i d i n the growth medium \ of P. aeruginosa. - 9 -From these data i t would appear that the enzyme enolase is not concerned with the formation of pyruvate by P. aeruginosa. This serves as further evidence that this organism does not dissimilate glucose by way of the conventional Embden-Meyerhof system. The quantitative determinations for pyruvic acid by the procedure of Friedemann and Haugen (ll) on the fluoride and non fluoride media showed an accumulation of pyruvate with the increasing age of the culture, suggesting a slowing down of the enzyme activity necessary for pyruvate oxidation, Figure 2 . This explanation was not confirmed when the cells were tested in the Warburg, for the 40 hour cells had a strong system for dissimilating pyruvate, Figure 1. P. aeruginosa was grown on compounds which could conceivably be oxidized without going through pyruvic acid. The carbon sources for this experiment were glycerol, n propyl alcohol, iso amyl alcohol and malohic acid. For controls glucose and pyruvic were used as growth substrates. The cells harvested from a l l of these media bad the ability to immediately oxidize pyruvate when tested in the Warburg respirometer. Since i t does not seem possible to grow P. aeruginosa deficient in pyruvic oxidase i t is not possible to make use of the technique of simultaneous adaptation for determining whether or not pyruvic is a metabolic intermediate. - 10 -DISCUSSION The t h e o r y o f s i m u l t a n e o u s a d a p t a t i o n c o u l d n o t be e m p l o y e d f o r d e t e r m i n i n g p y r u v a t e a s a m e t a b o l i c i n t e r m e d i a t e when g r o w i n g P . a e r u g i n o s a o n s u b s t r a t e d i s s i m i l a r i n s t r u c t u r e t o t h a t o f g l u c o s e a s c e l l s h a r v e s t e d f r o m t h e s e m e d i a were n e v e r d e f i c i e n t i n t h e p y r u v a t e o x i d i z i n g enzyme . C o n s i d e r a b l e d i f f i c u l t y was e x p e r i e n c e d w i t h t h e p r o c e d u r e o f C a v a l l a n i e t a l (4) f o r s e p a r a t i n g p y r u v i c h y d r a z o n e f r o m t h e c o n c e n t r a t e d m o t h e r l i q u o r . E t h y l a c e t a t e f o r m e d a n e m u l s i o n w i t h t h e c o n c e n t r a t e d f i l t r a t e a n d i t was t h e r e f o r e n e c e s s a r y t o f i n d a n o t h e r s o l v e n t e q u a l l y a s good f o r t h e e x t r a c t i o n o f p y r u v i c h y d r a z o n e . T o l u e n e was f o u n d t o be t h e most s u c c e s s f u l s o l v e n t . I n s u p p o r t o f t h i s o b s e r v a t i o n i s t h e w o r k o f F r i e d e m a n n a n d Haugen ( l l ) who u s e d a f i v e m i n u t e r e a c t i o n o f 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 i t h x t h e sample a n d t h e n e x t r a c t e d w i t h t o l u e n e . U s i n g t h i s p r o c e d u r e t h e y r e p o r t t h a t o n l y p y r u v i c a c i d r e a c t s w i t h t h e 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 i t h i n 5 m i n u t e s , o x a l a c e t a t e a n d «=< k e t o -g l u t a r a t e r e q u i r i n g 25-30 m i n u t e s f o r c o m p l e t e r e a c t i o n . The t w o R f v a l u e s f o r p y r u v i c h y d r a z o n e s w e r e i d e n t i f i e d a s t h e f r e e a c i d o f p y r u v i c h y d r a z o n e a n d t h e e t h y l e s t e r o f p y r u v i c h y d r a z o n e . T h e s e compounds w e r e i d e n t i f i e d by p r e p a r i n g d e r i v a t i v e s o f b o t h compounds a n d i h e n d e t e r m i n i n g t h e i r m e l t i n g p o i n t s . P y r u v i c a c i d h y d r a z o n e h a d a H i . P . o f 213°C. a n d t h e e t h y l e s t e r o f p y r u v i c a c i d h y d r a z o n e m e l t i n g p o i n t was 155°C F r o m t h e d a t a p r e s e n t e d i t c a n be c o n c l u d e d t h a t p y r u v i c a c i d i s a compound t h r o u g h w h i c h t h e o x i d a t i o n o f g l u c o s e b y P . a e r u g i n o s a p r o c e e d s . - 11 -SUMMARY 1. C e l l s h a r v e s t e d f r o m g l u c o s e m i n e r a l m e d i a a t 16, 28 a n d 4-0 h o u r s were f o u n d t o have a n u n i m p a i r e d s y s t e m f o r o x i d i z i n g p y r u v a t e . 2 . P y r u v i c a c i d was i s o l a t e d f r o m a c o n c e n t r a t e d g r o w t h f i l t r a t e , a s t h e 2 - 4 d i n i t r o p h e n y l h y d r a z o n e d e r i v a t i v e a n d i d e n t i f i e d b y means o f p a p e r c h r o m a t o g r a p h y . 3. P y r u v i c a c i d was shown t o be a n i n t e r m e d i a t e i n t h e o x i d a t i o n o f g l u c o s e . - 12 -PART II. THE ISOLATION OF GLUCOSE OXIDASE AND A STUDY OF ITS PROPERTIES Glucose Aerodehvdrogenase Glucose oxidase was discovered by Muller (23) in Aspergillus niger and Penicilium glaucum. and was reinvestigated by him in 1940 (2A). He found that the enzyme catalyzes the oxidation of glucose to gluconic acid by means of molecular oxygen and that i t oxidizes mannose and galactose at a slower rate but does not attack other sugars. The activity was not affected by cyanide. Harrison (12), working with ox liver tested the inhibiting effect of d. glucose on the oxidation of hexose diphosphate ester. Due to the similarity in structure of glucose and the phosphate ester he expected d. glucose to be absorbed on the surface of the enzyme, thus inhibiting its reaction on the hexose diphosphate. Instead of inhibition a marked acceleration in oxygen uptake was observed. He found that the liver preparation contained a dehydrogenase capable of oxidizing glucose to gluconic acid. The enzyme was water soluble, and required cozymase to function. He showed that methylene blue and the cytochrome system could serve as carriers. Ogston and Green (27) were unable to verify Harrison'B results with the cytochrome system but were able to show that yeast flavoprotein could serve as the carrier in the aerobic oxidation of d. glucose. Hawthorne and Harrison (lA) showed that the cytochrome system of Keilin and Hartree catalyzed the oxidation of d. glucose and suggested that flavoprotein may be necessary for the f u l l activity of their dehydrogenase system. Franke and Lorenz (10) and Franke and Deffner (9) reinvestigated M'ullers enzyme employing a more purified preparation and demonstrated that during the oxidation of glucose to gluconic acid, oxygen was reduced - 13 -to hydrogen peroxide. They found also a proportionality between the activity of enzyme and its flavin content, and concluded that the enzyme must be a flavoprotein. More recently Coulthard, Michaelis, Short, Sykes, Skrimshire, Standfast, Brinkinshaw, Raistrick, (5) isolated an anti-bacterial glucose aerodehydrogenase from Penicillium notatum while investigating the antibacterial properties developed in a culture medium of the organism. The substance, notatin, was identified by them as glucose oxidase (6), and i t was shown that the antibacterial property was due to hydrogen peroxide formed in the presence of glucose. They established its flavoprotein nature and suggested the prosthetic group might be alloxazine adenine dinucleotide. Keilin and Hartree (1$) using an amino acid oxidase system for testing the activity of the prosthetic group of notatin found i t to be alloxazine adenine dinucleotide as suggested by Coulthard et al (6). Eichel and Wainio (8) reinvestigating Harrison's work using lambs liver as the source of their enzyme verified Harrison's (12) indophenol oxidase system as the necessary carrier and showed that the lack of increased activity of Ogston and Green's (27) system was due to the high cytochrome c in the cytochrome oxidase preparation. They showed as did Ogston and Green (27) that flavoprotein could mediate the reaction, the activity, however, being less that that of the cytochrome oxidase system. Interest in glucose dehydrogenase was stimulated when Norris and Campbell (26) identified gluconic and 2 ketogluconic acids by paper partition chromatography, as the breakdown products of glucose dissimilation by the aerobic organism P. aeruginosa. Later Stokes and Campbell (32) using sloppy dried cells were able to stop glucose breakdown at 2 keto-- 1A -gluconic, the second .intermediate in the oxidation of glucose by P. aeruginosa. Using their method of preparing sloppy dried cells experiments were conducted to isolate the reaction, glucose to gluconic acid, the first 6tep of glucose degradation by the organism P. aeruginosa, and to study some of the characteristics of the enzyme responsible for the production of gluconic acid from glucose. - 15 -METHODS Bacteriological The methods of cultivation of Pseudomonas aeruginosa 9027 were the same as employed under Methods Part I. Whole cells used for Thunberg experiments were standardized using the electrophotometer as employed under Methods Part I. A dried cell preparation was obtained by inoculating a 24 hour culture Into 15 liters of glucose mineral media, incubating for a period of 24 hours at 30°C. and then harvesting the cells with a Sharpies centrifuge. The cells thus obtained, were washed with 150 mis of H/l5 phosphate buffer at pH7.2, recentrifuged and then taken up in 80 mis of distilled water, spread in a thin film on a petrie dish and dried in vacuo over phosphorus pentoxide. Time required for drying of the cells ranged between 38 and 48 hours. A conventional Warburg apparatus was used to follow the oxygen uptake of substrate oxidation by the dried cell suspensions. In the studies of the influence of inhibiting substances on the oxidation of glucose, the cell preparation was incubated 30 minutes with the inhibitor prior to the addition of substrate from the side arm. The Warburg cups contained 1.5 ml of Sorenson's phosphate buffer pfl.7.5 and 0.5 ml cells in the main compartment. Substrates to be tested were tipped in from the side arm. The center well contained 0.15 ml of 20% K0H for the absorption of carbon dioxide. The total volume of fluid in the cups was 3*15 ml., (0.5 ml of added substrate xvas equal to 54uM. of oxygen.) The Thunberg technique was employed for the study of the anaerobic dehydrogenase activity of P. aeruginosa cells, using methylene blue to mediate the reaction. The Thunberg tubes were evacuated 3 minutes prior - 16 -to closing off, and then placed in a 37°C. water bath for 10 minutes before the substrate was tipped from the side arm. Reactions were allowed to proceed to 90% reduction of methylene blue. Chemical The descending method of paper chromatography was used for the identification of the end products of Warburg experiments. Unknowns to be tested were removed from the Warburg cups at the completion of an experiment, and were introduced onto Whatman #1 f i l t e r paper by means of fine capillary tubes. The f i l t e r paper was then hung from a trough at the top of a glass chamber and the atmosphere was then saturated with the solvent vapor prior to running of the chromatogram. The solvent used was methyl alcohol 4-5%, ethyl alcohol 45%, water 10%. The chromatogram was irrigated for a period of 15-18 hours, the paper was then dried in air, and sprayed with 0.1N silver nitrate in 5N ammonium hydroxide. The sprayed sheet was then dried in a semi dark room or in the absence of direct sunlight. This particular chromatographic procedure was used by Norris et a l (26), for the identifications of intermediates in the metabolism of glucose by P. aeruginosa. -17 -EXPERIMENTAL Anaerobic Studies The Thunberg method for determining dehydrogenase reactions in the presence of methylene blue has only limited value with P. aeruginosa due to the high endogenous activity of the cells. In order that the endogenous activity might be:reduced or eliminated entirely, various techniques were employed. When aeration as used in the starvation of resting cells was employed (Quastel and Whetham, 30) for a period of A hours the aerated cells exhibited normal endogenous activity. A young culture of 8 hours as proposed by Osten (28) was also used employing two different concentrations of cells and methylene blue. The concentrations of methylene blue at 1:1500 and the 20X cells (cells concentrated 20 times), showed a satisfactory reduction time for endogenous activity, but proved unsatisfactory for studies of glucose dehydrogenation. Table 2. However, the results do indicate dehydrogenase activity in the anaerobic dissimilation of glucose, Table 2. Although the conditions for the study of glucose dehydrogenase by this procedure were not entirely satisfactory attemps to improve the method were studied employing glucose at different pfls. It was found that a glucose concentration of 0.Q2M was most satisfactory for the dehydrogenase studies, and using this concentration of substrate in the Thunberg tubes experiments on the different pfl's were carried out. Results expressed in Table 3 were from cells harvested after 14 hours. Cells represented in Table 2 were harvested after 8 hours. It can be seen that the 14 hour cells at pH6.0 are equivalent to 8 hour cells at pH7.0, at the same concentration of cells and methylene blue. A - 18 -TABLE 2 THE REDUCTION OF METHYLENE BLUE BY 8 HOUR WHOLE CELLS Concentration of Cells Substrate Reduction Times MB 1:1500 MB 1:2500 20X Endogenous 68 min. 20 min. Glucose 39 min. • 15 min. 40X Endogenous 26 min. 1 4 min. Glucose 21 min. 12"-min7 Thunberg tubes contained 0 . 5 c.c. of M/l5 phosphate buffer pH7.0, 1 c c . of cell suspension. 0 . 5 c.c. of methylene blue, 0 . 5 c.c. of 0 . 0 2 M glucose. Final volume Of 3 . 0 c.c. with distilled water. - 19 -TABLE 3 METHYLENE BLUE REDUCTION BY U HOUR OLD WTOT.F. CTT.T.S PH Substrate Reduction Time 7.5 Endogenous Glucose 30 min. 27 min. 7.0 Endogenous Glucose 30 min. 20 min. 6.5 Endogenous Glucose 29 min. 23 min. 6.0 Endogenous Glucose 64 min. 26 min. Thunberg tubes contained 1.0 c c . of 20X cell suspension, 0.5 c c M/l5 phosphate buffer, 0.5 c c 0.02M glucose, 0.5 c c 1:1500 methylene blue. Final volume 3«0 c.c - 20 -comparison between results in Table 2 and 3 at pH7.0 could not be drawn effectively because of this time variation. However, Table 3 shows a slight increased time between the endogenous and glucose activities as the pH decreases. A pH of 7.0 was found to give the shortest reduction time for glucose indicating this pH may be approaching the optimum. From the above results i t is noted that an incubation period of 8 hours or less for P. aeruginosa is to be favored for anaerobic studies. The Thunberg technique was found however, to have limited use for dehydrogenase studies when employing whole cells of P. aeruginosa. The unsatisfactory results obtained with whole cells of P. aeruginosa led to experimental work seeking a selective method for rupturing the cell membrane or increasing permeability without destroying the enzyme activity of the cel l . Acetone treatment was first tried as a means of increasing cell permeability. Whole cells were treated with ice cold acetone, one volume of cell suspension, 40X, to two volumes of acetone. No activity was recorded from these acetone treated cells when tested in the Thunberg tubes. In an effort to rupture the cell membrane and liberate the cell enzymes, freezing and thawing of the cells in a 17% saline solution, using dry ice and acetone as the freezing mixture, was employed. The cell suspension obtained by this method showed no activity when diluted to a concentration of 0*5% saline and tested in Thunberg tubes. Grinding the cells with an ice cold pestle and mortar with the aid of fine silica also destroyed cell activity. A dried cell preparation as described previously was also found to - 21 -be without glucose dehydrogenase activity when tested in the Thunberg tubes, Table A. However, Stokes and Campbell (32) using the same type of cell preparation for Warburg studies showed that glucose was oxidized to 2 ketogluconic.acid. The same type of cell preparation as used by Stokes and Campbell was expected to give the deBired dehydrogenase activity when tested anaerobically in the presence of methylene blue because of the high glucose d i s s i m i l a t i n g activity that was observed in Warburg experiments by these workers. However, i t was found glucose was not dissimilated under anaerobic conditions by the cell preparation as evidenced by the lack of methylene blue reduction, Table A. When testing these same cells in the Warburg apparatus the oxidation of glucose was observed, thus confirming the findings of Stokes and Campbell (32) by this organism. These results were the reverse of ^ arrison's (12) who, using an ox liver enzyme preparation showed that no oxygen was taken up by the enzyme plus substrate when tested in the Barcroft respirometer but found methylene blue could mediate this reaction. He showed later (13) that in the presence of cytochrome-indo-phenol oxidase system, oxygen was taken up in the presence of glucose by the enzyme with which he was working. In addition to glucose, gluconic, 2 ketogluconic and succinic acids were tested, Table A. Gluconic and succinic acids were two substrates for which the cell preparation possessed dehydrogenases. The reduction of methylene blue in the presence of these two substrates by these non proliferating cells is evidence that these two compounds are intermediates in the carbohydrate metabolism of P. aeruginosa, the enzyme being present in the dried cells as shown by the rapid reduction time of five minutes. - 22 -TABLE A METHYLENE BLUE REDUCTION BY DRIED CELL PREPARATION S u b s t r a t e R e d u c t i o n T ime G l u c o s e 20 m i n s . G l u c o n i c 5 m i n s * 2 K e t o g l u c o n i c 20 m i n s . S u c c i n i c 5 m i n s . Endogenous ( n o s u b s t r a t e ) 20 m i n s . T h u n b e r g t u b e s c o n t a i n e d 0.5 c . c . c e l l s u s p e n s i o n c o n t a i n i n g 20 mgs . o f c e l l s , 0.5 c . c . o f 1:1500 m e t h y l e n e b l u e , 0.5 c . c . o f M/l5 p h o s p h a t e b u f f e r pH7.2, 1.0 ' c . c . o f 0.015M s u b s t r a t e . F i n a l v o l u m e was made t o 3.0 c . c . w i t h d i s t i l l e d w a t e r . The r e a c t i o n was t a k e n t o 90% r e d u c t i o n o f m e t h y l e n e b l u e . - 23 -From this work i t may be concluded that the glucose enzyme in cell preparation of P. aeruginosa does not function under anaerobic conditions, or that methylene blue cannot mediate the reaction for glucose oxidase in the presence of glucose as the substrate. Aerobic Experiments Sloppy dried cells, grown for a 2A hour period and dried over phosphorus pentoxide in vacuo, were tested in the Warburg respirqmeter on various substrates in order to determine i f more than one compound was oxidized under aerobic conditions by the cell preparation. Glucose, gluconate, succinate, fumarate, pyruvate, 2 ketogluconate, e*c ketoglutarate, citrate, iso citrate, aconitate and acetate were the substrates employed at!pH7.2. Of these energy sources only glucose and succinic acid showed any reasonable oxygen uptake at pH7.2. Measurable activity for gluconic acid was also observed. Further work in the problem was in large measure confined to a detailed study of the oxidation of glucose. The glucose activity of the cell preparation having been established, the next step was to isolate the specific reaction, glucose to gluconic acid the f i r s t intermediate in the dissimilation of glucose by the organism under study. Stokes and Campbell (32) were able to accumulate 2 ketogluconic acid, when testing their cell preparation on glucose at pH7.2. By varying the pH i t was found possible to stop glucose oxidation at gluconic acid. With the preparation under study i t was found by increasing the pfl range between pH?.5 to 8.0, glucose oxidation was complete at gluconic acid, as evidenced by oxygen uptake, Figure 3• Further con-firmation of this finding is presented in Figure A. The results obtained by a chromatographic analysis of the working substrates are given in T h e o r e t i c a l o x y g e n u p t a k e f o r t h e o x i d a t i o n o f g l u c o s e t o g l u c o n i c a c i d was 1 0 0 . 8 m i c r o l i t e r s a n d f o r t h e o x i d a t i o n o f g l u c o s e t o 2 k e t o g l u c o n i c a c i d was 2 0 1 . 6 m i c r o l i t e r s . MINUTES F i g . 4. The e f f e c t o f pH o n t h e o x i d a t i o n o f g l u c o s e a n d g l u c o n i c a c i d by a d r i e d c e l l p r e p a r a t i o n o f P . a e r u g i n o s a . T h e o r e t i c a l o x y g e n u p t a k e f o r complete o x i d a t i o n o f g l u c o s e t o g l u c o n i c a c i d was 1 0 0 . 8 m i c r o l i t e r s , f o r g l u c o n i c a c i d t o 2 k e t o g l u c o n i c a c i d was 1 0 9 . 8 m i c r o l i t e r s and f o r g l u c o s e t o 2 k e t o g l u c o n i c a c i d was 2 0 1 . 6 m i c r o -l i t e r s . * • * - g l u c o s e o x i d a t i o n — o = g l u c o n i c a c i d o x i d a t i o n - 26 -Table 5> Rf values for glucose, gluconic acid and 2 ketogluconic acid are indicated. At pH7.5 oxygen uptake for glucose at 140 minutes is 109 uls. and for gluconic acid 18 uls. The theoretical oxygen uptake for glucose to gluconate is 100.8 uls. and for gluconic acid to 2 ketogluconate is 109.7 uls. TABLE 5 CHROMATOGRAPHIC ANALYSIS OF WARBURG SUBSTRATES Rf Color on fi r s t developing later turning black Glucose • 58 Gluconic .25 red brown spot 2 Ketogluconic .295 grey spot Product of oxidation from glucose pH7.0 .28 greyish spot from gluconic pH7.0 .29 greyish spot from glucose pH7.5 .26 red-brown spot from gluconic pH7.5 .26 red brown spot Consideration of the chromatographic findings recorded in Table 5, strengthen the conclusions reached from a study of the results recorded in Figures 3 and 4» The product of oxidation of glucose at pH7.5 would appear to be gluconic acid further strengthening the suggestion that the reaction at p^ 7.5 is concerned with the transformation of glucose to gluconic acid, while at lower pH7.0, 2 ketogluconic was found to be the end product, a finding recorded in Figure A. At pH7«0 the oxygen uptake was found to be 192 uls. and the theoretical oxygen uptake for this reaction is 201.6 uls. It_can be seen from Figure 4 that gluconate is oxidized rapidly at pH6.5, more slowly at pH7.0, and not at a l l at pH7.5. With - 27 -this strong evidence and confirmation of glucose oxidation to gluconic acid as recorded in Table 5 at pH7.5, i t was concluded that further studies with cell preparation at pH7.5 under the same conditions, would yield gluconic acid. Based on this assumption descending chromatographs were discontinued except when testing a fresh cell preparation. A second cell preparation showed essentially the same type of curves for glucose oxidation except that a pH of 8.G was required in order to establish the glucose to gluconic acid reaction. The pH experiments on glucose and gluconic acid oxidation indicate that the enzyme responsible for the reaction glucose to gluconic acid functions at a higher pfi than does the enzyme required to convert gluconic to 2 ketogluconic acid. Whatever the mechanism concerned with this physical blocking of the two enzymes at the higher and lower pHs i t apparently does not function at pH7.0 as evidenced by the oxygen uptake recorded in Figures 3 and A. Enzyme Action In an attempt to determine the influence of various proteolytic enzyme properties on glucose oxidase the following experiments were carried out. Trypsin, at pH7.5> completely inactivated the glucose enzyme, when incubated at room temperature for a period of 8 hours. A control (no trypsin) at pH7.5 and at room temperature for 8 hours showed normal oxygen uptake at the end of 140 minutes. Takadiastase at pH6.0 and under the same conditions as trypsin showed no signs of inactivating glucose oxidase. The action of papain and pepsin at pH3«5 could not be determined as the low pH destroyed the activity of the dried cell preparation. These findings are contrary to those recorded by Coulthard - 28 -at al (6) who showed that neither pepsin nor trypsin inactivated glucose aerodehydrogenase of Penicilllum notaturn. Their enzyme showed a stability range from pH3.5 to pfi6.0 and pepsin at pH2.5 for 8 hours did not destroy the glucose oxidizing power of their enzyme. Effect of Light In studies on the influence of light on glucose oxidase the method employed for the microbiological assay of riboflavin was adopted, i.e. exposure to a strong light, 100 watts for a period of eight to ten hours. As riboflavin is a flavoprotein and destroyed by strong light i t was expected that i f the protein of glucose oxidase was a flavin, then, exposure to a 100 watt lamp for eight to ten hours would destroy the enzyme action. The dried cell preparation was divided into two portions. One portion of the preparation was subjected to strong light in an ice bath, a second portion, serving as a control, was stored in the refrig-erator. A sample of the cells exposed to light was removed after eight hours, while samples of the control were removed at zero and eight hours. The action of light on the ability of the cell preparation to oxidize glucose was determined. The influence of added riboflavin on both normal and light treated cells was also studied. The results of these experiments are recorded in Figure 5. From these data i t can be seen that some significant decrease in the ability of the cell preparation to oxidize glucose after exposure to light for eight hours is shown. The slight increase in activity on the addition of riboflavin to the non treated cells may be attributed to some oxidation of riboflavin by the non treated cells. That the system was partially destroyed by strong light is some - 29--200 to cc U J j -_l o or o Id <t fclOO U J o >-X o MINUTES F i g . 5 * The e f f e c t o f s t r o n g l i g h t and r i b o f l a v i n o n a d r i e d c e l l pre—-p a r a t i o n o f P . a e r u g i n o s a . C u r v e s 1 = c e l l p r e p a r a t i o n a t 0 h o u r s . C u r v e s 2 = c e l l p r e p a r a t i o n h e l d i n t h e r e f r i g e r a t o r 8 h o u r s . C u r v e s 3 - c e l l p r e p a r a t i o n e x p o s e d t o 100 w a t t l a m p f o r 8 h o u r s . - o g l u c o s e - x g l u c o s e •»• r i b o f l a v i n (3mgms.) - 30 -i n d i c a t i o n t h a t the enzyme may be a f l a v i n . The f a i l u r e o f r i b o f l a v i n t o r e s t o r e the enzyme 1 s a c t i v i t y would i n d i c a t e t h a t r i b o f l a v i n i s n o t concerned w i t h g lucose o x i d a s e o f P . a e r u g i n o s a . E f f e c t o f Temperature I n o r d e r t o determine the heat l a b i l i t y o f g lucose ox idase a t pH7.5, samples o f the c e l l s were heated i n d i s t i l l e d water f o r p e r i o d s o f 5, 10, 30 and 60 minutes a t temperatures o f 37°C, 45°C. and 55°c« r e s p e c t i v e l y . I t had been found e a r l i e r t h a t c e l l s h e l d f o r 8 h o u r s , ( s t u d i e s o f p r o t e o l y t i c enzyme a c t i o n on the c e l l p r e p a r a t i o n ) , a t room temperature i n phosphate b u f f e r , pH7.5, showed l i t t l e s i g n o f d e t e r i o r a t i o n . The temperature o f 55°C. was found t o d e s t r o y the enzyme a t a l l p e r i o d s . The enzyme p r e p a r a t i o n a f t e r 30 minutes a t A5°0* and 60 minutes a t 37°C. was p a r t i a l l y i n a c t i v a t e d , as evidenced by the r a t e and t o t a l oxygen uptake when t e s t e d i n the Warburg r e s p i r o m e t e r . The r e s u l t s r e c o r d e d here i n d i c a t e g lucose o x i d a s e i s s e n s i t i v e t o a h i g h temperature f o r a s h o r t p e r i o d o f t ime and i s t h e r e f o r e c o n s i d e r e d t o be heat l a b i l e . A c t i o n o f I n h i b i t o r s I n the c h a r a c t e r i z a t i o n o f an enzyme, i n h i b i t o r s are used t o determine c e r t a i n p r o p e r t i e s o f the enzyme and i t s c o - f a c t o r s . Sodium a z i d e and potass ium cyanide a r e two such compounds and t h e i r s p e c i f i c r e a c t i o n i s concerned w i t h i n h i b i t i o n o f r e s p i r a t o r y enzyme a c t i o n . Sodium a z i d e was used a t a c o n c e n t r a t i o n o f 2xl0"*3l a t pH7.5 and pH6.5. A t pH7.5 sodium a z i d e i n h i b i t e d the o x i d a t i o n o f g lucose a lmost 50% w h i l e a t pH6.5 i n h i b i t i o n was complete . A t pH6.5 w i t h sodium a z i d e - 31 -t h e o x i d a t i o n o f g l u c o n i c a c i d was a l s o c o m p l e t e l y i n h i b i t e d . P o t a s s i u m c y a n i d e , a compound u s e d i n d e t e r m i n i n g w h e t h e r o r n o t t h e c y t o c h r o m e s a r e r e q u i r e d b y t h e enzyme s y s t e m t o f u n c t i o n p r o p e r l y , c a u s e d c o m p l e t e i n h i b i t i o n o f g l u c o s e o x i d a t i o n d u r i n g t h e f i r s t AO m i n u t e s a f t e r w h i c h t i m e t h e c e l l p r e p a r a t i o n began t o t a k e up o x y g e n . I t was as sumed t h a t t h i s c o n d i t i o n was c a u s e d b y t h e d i s t i l l i n g o f c y a n i d e t o t h e a l k a l i n e c e n t e r w e l l w h i c h p r o b a b l y was n o t p r o p e r l y s a t u r a t e d w i t h p o t a s s i u m c y a n i d e . On f u r t h e r e x p e r i m e n t a t i o n i t was f o u n d t h a t no c a r b o n d i o x i d e was e v o l v e d when g l u c o s e i s o x i d i z e d b y t h e c e l l p r e p a r a t i o n , a f i n d i n g t o be e x p e c t e d u p o n o x i d a t i o n o f g l u c o s e t o g l u c o n i c a c i d . F o r t h i s r e a s o n t h e e f f e c t o f p o t a s s i u m c y a n i d e on t h e o x i d a t i o n o f g l u c o s e c o u l d be t e s t e d w i t h o u t p o t a s s i u m h y d r o x i d e i n t h e c e n t e r w e l l . The r e s u l t s o f e x p e r i m e n t s c o n d u c t e d u n d e r t h e above c o n d i t i o n s w i t h t h e n e c e s s a r y c o n t r o l s a r e r e c o r d e d i n T a b l e .6* C y a n i d e i n t e r f e r e s w i t h t h e o x i d a t i o n o f g l u c o s e b y t h e d r i e d c e l l s a t t h e two c o n c e n t r a t i o n s u s e d . V a r i o u s w o r k e r s (9), (10), (6) h a v e shown t h a t when g l u c o s e i s o x i d i z e d t o g l u c o n i c a c i d , h y d r o g e n p e r o x i d e i s f o r m e d . S i n c e t h e c e l l s w e r e f o u n d t o h a v e a h i g h c a t a l a s e a c t i v i t y , a c y a n i d e s e n s i t i v e enzyme t h a t d e s t r o y s h y d r o g e n p e r o x i d e , i t was c o n c l u d e d t h a t t h e c y a h i d e c o u l d f u n c t i o n i n two c a p a c i t i e s j o n e , i t m i g h t i n h i b i t c a t a l a s e t h u s a l l o w i n g p e r o x i d e t o a c c u m u l a t e i n w h i c h c a s e i t m i g h t be t o x i c t o t h e o x i d a t i o n o f g l u c o s e a n d , t w o , i n t e r f e r i n g w i t h c y t o c h r o m e w h i c h m i g h t be r e q u i r e d t o m e d i a t e t h e o x i d a t i o n o f g l u c o s e . I n o r d e r t o t e s t t h e f o r m e r h y p o t h e s i s t h e m e t h o d o f M a i n and S h i n a (21) was u s e d . N e g a t i v e r e s u l t s w e r e o b t a i n e d u s i n g t h i s p r o c e d u r e t h u s i n d i c a t i n g a c y a n i d e s e n s a t i v e enzyme w i t h o u t t h e p r o d u c t i o n o f h y d r o g e n p e r o x i d e . O t h e r e n z y j i e i n h i b i t o r y a g e n t s were, a l s o s t u d i e d . R e s u l t s o f t h i s - 32 -TABLE 6 Oxidation of Glucose in the Presence and Absence of _ KOH and Cyanide After 140 Minutes Substrate Oxygen Uptake in uls. Center Well Glucose 99 No KOH Glucose 102 KOH Glucose *10~% KCN 96 uls. KOH Glucose *-10~% KCN 51 uls. No KOH Glucose r l O - % KCN No oxygen uptake KOH Glucose «-10~2M KCN No oxygen uptake No KOH Warburg cups contained" 0.5 c.c. cell preparation, 1.5 c.c. of M/15 phosphate buffer pH7.5, 0.5 c.c. glucose, 0-3 c.c. inhibitor, made to final volume of 3.0 c.c. with distilled water. Theoretical oxygen uptake is 100.8 uls. - 33 -study are recorded in Table 7.) Malonate interferes with the succinate-fumarate system and since as shown in Table 7 no inhibition was observed when the compound was employed i t is to be concluded that the succinate-fumarate system is not linked directly to the oxidation of glucose by the dried cells. Iodoacetate, a chemical employed for detecting sulfhydryl groups, shows no inhibition and therefore shows that glucose 'oxidase does not contain a sulfhydryl grouping. These results are in agreement with Harrison (12) and also Eichel and Wainio (8). Information on a specific reaction for arsenate is obscure, however Pickett and Clifton (29) testing the effect of arsenate on mocroorganisms showed that both pyruvate and glucose oxidation were blocked by arsenate. With the enzyme under study no such inhibition was observed. TABLE 7 INHIBITORS FURTHER CHARACTERIZING THE ENZYME Inhibitor Reaction Arsenate .86x10M No inhibition Arsenate 1.72xlO""M No inhibition Malonate .05M No inhibition Iodoacetate .002M No inhibition - 3A -Coenzyme I n a s t u d y o f t h e coenzyme o f g l u c o s e a e r o d e h y d r o g e n a s e many methods f o r s e p a r a t i n g t h e apoenzyme f r o m t h e coenzyme were s t u d i e d i n a n a t t e m p t t o f i n d a method t h a t w o u l d y i e l d two f r a c t i o n s n e i t h e r o f w h i c h w o u l d p o s s e s s a c t i v i t y b u t when c o m b i n e d , w o u l d be r e s t o r e d t o f u l l a c t i v i t y . Yifhen t h e c o n v e n t i o n a l method f o r s e p a r a t i n g coenzyme f r o m i t s p r o s t h e t i c g r o u p , n a m e l y , d i a l y s i s a g a i n s t d i s t i l l e d w a t e r f o r a p e r i o d o f 8 h o u r s o r more ( u s e d b y many w o r k e r s i n t h e f i e l d o f enzyme c h e m i s t r y ) f a i l e d o t h e r methods c i t e d i n t h e l i t e r a t u r e were t r i e d . T h e o r e l l (35) p r o p o s e d d i a l y s i s a g a i n s t 0.02N HC1 a s a method o f r e m o v i n g coenzyme f r o m f l a v o p r o t e i n . S t r a u b (33) u s i n g a c e t a t e b u f f e r p H A . 6 p r e c i p i t a t e d e x c e s s p r o t e i n w i t h a l c o h o l i c ammonium s u l p h a t e , c e n t r i f u g e d t h e i n s o l u b l e p r o t e i n a n d a b s o r b e d t h e f l a v o p r o t e i n o n a l u m i n a g e l (6 y), e l u t e d t h i s w i t h d i s o d i u m h y d r o g e n p h o s p h a t e and d i a l y z e d . W a r b u r g a n d C h r i s t i a n (36) s | > l i t t h e o l d y e l l o w enzyme i n t o coenzyme a n d apoenzyme b y t r e a t i n g t h e enzyme w i t h s a t u r a t e d ammonium s u l p h a t e and t h e n s t i r r i n g i n t o 0.1N HC1. The f l a v o p r o t e i n r e m a i n e d i n s o l u t i o n , w h i l e t h e coenzyme was p r e c i p i t a t e d . T h e s e methods a l t h o u g h s a t i s f a c t o r y t o t h e w o r k e r s l i s t e d above f o r s e p a r a t i n g f l a v o p r o t e i n f r o m i t s coenzyme , were f o u n d t o be u n s a t i s f a c t o r y f o r t h e P . a e r u g i n o s a c e l l p r e p a r a t i o n . M o d i f i c a t i o n s o f t h e above p r o c e d u r e s were t r i e d w i t h o u t s u c c e s s . H o w e v e r , t h e method o f W a r b u r g and C h r i s t i a n d i d h e l p i n f i n a l l y e v o l v i n g a p r o c e d u r e t h a t was s a t i s f a c t o r y i n s e p a r a t i n g t h e c e l l p r e p a r a t i o n i n t o two f r a c t i o n s . - 35 -P r o c e d u r e f o r S e p a r a t i n g G l u c o s e O x i d a s e i n t o Two F r a c t i o n s A known w e i g h t o f c e l l s , 500 mgms. was s u s p e n d e d i n 30 c . c . o f d i s t i l l e d w a t e r a n d m i x e d u n t i l t h e s u s p e n s i o n was homogeneous . To t h i s p r e p a r a t i o n 2A .6 gms. o f ammonium s u l p h a t e were a d d e d , w i t h c o n t i n u e d s t i r r i n g o v e r a p e r i o d o f 1 5 - 2 0 m i n u t e s . A c o a g u l u m o f p r o t e i n s l o w l y f o r m e d a t t h e s u r f a c e o f t h e t r a n s p a r e n t l i q u o r . The two f r a c t i o n s were t h e n s e p a r a t e d b y c e n t r i f u g a t i o n . B o t h f r a c t i o n s were t h e n p l a c e d i n s e p a r a t e s a u s a g e w r a p p i n g s a n d d i a l y z e d o v e r n i g h t ( 1 6 h o u r s ) , i n t h e r e f r i g e r a t o r , a g a i n s t d i s t i l l e d w a t e r , t o remove t h e ammonium s u l p h a t e . The p r e c i p i t e d m a t e r i a l was t h e n made t o s u c h a v o l u m e t h a t 1 c . c . c o n t a i n e d 20 mgms o f m a t e r i a l . The r e s u l t s o f t h i s e x p e r i m e n t a r e r e c o r d e d i n F i g u r e 6. I t c a n be s e e n t h a t t h e two f r a c t i o n s a r e w i t h o u t t h e n o r m a l a c t i v i t y o f t h e d r i e d c e l l p r e p a r a t i o n , a s shown i n F i g u r e 1 . F r a c t i o n 1 showed no a c t i v i t y , F r a c t i o n 2 , s l i g h t a c t i v i t y and F r a c t i o n s 1 and 2 c o m b i n e d , o n l y s l i g h t a c t i v i t y . H o w e v e r , when F r a c t i o n 1 , ( t h e c o a g u l u m o f ammonium s u l p h a t e t r e a t m e n t ) , was c o m b i n e d w i t h magnes ium a t 1 0 ~ % c o n c e n t r a t i o n a t h e o r e t i c a l o x y g e n u p t a k e a t t h e end o f 40 m i n u t e s was o b s e r v e d ( g l u c o s e t o g l u c o n i c 100 .8 u l s ) . I t was a l s o f o u n d t h a t magnesium a t 1 0 ~ % c o n c e n t r a t i o n c a n s u b s t i t u t e f o r magnes ium, h o w e v e r t h e r a t e o f a c t i v i t y a t t h e pH e m p l o y e d was n o t a s g r e a t , a l t h o u g h t h e f i n a l o x y g e n u p t a k e was g r e a t e r . F r a c t i o n 2 , ( t h e e f f l u e n t o f t h e ammonium s u l p h a t e t r e a t m e n t ) , was n o t s t i m u l a t e d b y t h e a d d i t i o n o f e i t h e r i o n i W i t h t h e s e p a r a t i o n o f t h e enzyme i n t o two f r a c t i o n s e x p e r i m e n t s were c o n d u c t e d t o e s t a b l i s h t h e s p e c i f i c coenzyme a n d s u b s t i t u t i n g c o f a c t o r s . Coenzyme 1, a d e n o s i n e t r i p h o s p h a t e , c y t o c h r o m e c , a n d members o f t h e - 36 -to ce UJ _ J o CC O 200 UJ 0. Z U l o >-X o 100 80 MINUTES F i g . 6 . R e a c t i v a t i o n o f c e l l p r e p a r a t i o n . T h e o r e t i c a l o x y g e n u p t a k e f o r t h e o x i d a t i o n o f g l u c o s e t o g l u c o n i c a c i d was 100.8 m i c r o l i t e r s . F l = F r a c t i o n 1 F2 = F r a c t i o n 2 Mg = Magnes ium 10"% + f r a c t i o n 1 Mn = Manganese 10~M& * f r a c t i o n 1 - 37 -v i t a m i n B c o m p l e x ; b i o t i n , n i a c i n , r i b o f l a v i n , p a n t o t h e n i c a c i d , t h i a m i n e , f o l i c a c i d , p y r i d o x a l , p a r a a m i n o b e n z o i c a c i d , were t r i e d . C o m b i n a t i o n s o f B c o m p l e x w i t h a n d w i t h o u t magnesium were a l s o u s e d . O f t h e s u b s t a n c e s t e s t e d o n l y t h e c o m b i n a t i o n s o f t h e v i t a m i n B c o m p l e x w i t h added magnesium showed r a p i d o x y g e n u p t a k e , a l w a y s s h o w i n g t h e same t y p e o f c u r v e as magnes ium a l o n e b u t n e v e r a s a c t i v e . I n a d d i t i o n t o t h e compounds —3 m e n t i o n e d manganese was a l s o u s e d i n a g r e a t e r c o n c e n t r a t i o n , 10~^M. H o w e v e r , t h e c u r v e o b t a i n e d u n d e r t h e s e c o n d i t i o n s was t h e same a s i n F i g u r e 6. ( F r a c t i o n 1 * M n 1 0 " % ) . F r o m t h e s e l i m i t e d e x p e r i m e n t s i t w o u l d a p p e a r t h a t magnes ium a c t s a s t h e coenzyme b u t c a n be r e p l a c e d by manganese . - 38 -DISCUSSION I n t h e s t u d y o f t h e p r o p e r t i e s o f g l u c o s e o x i d a s e a. c o m p a r i s o n may be d r a w n w i t h r e l a t e d enzyme s y s t e m s as r e c o r d e d i n t h e l i t e r a t u r e b y o t h e r w o r k e r s . I t must be remembered , h o w e v e r , t h a t i n a g e n e r a l c o m p a r i s o n one must c o n s i d e r t h e d i f f e r e n c e i n t h e s p e c i f i c i t y o f t h e p r o t e i n . A l t h o u g h t h e enzymes may c a t a l y z e t h e same r e a c t i o n , ( g l u c o s e o x i d a s e o f mic roorgan i sm, . , a n d g l u c o s e o x i d a s e o f mammalian t i s s u e ) , t h e i r p r o p e r t i e s may n o t be a p p l i c a b l e t o e a c h o t h e r due t o t h e d i f f e r e n c e i n t h e n a t u r e o f t h e p r o t e i n ( a p o e n z y m e ) . A n o t h e r d i f f i c u l t y e n c o u n t e r e d i s t h e i s o l a t i o n and p u r i f i c a t i o n o f t h e enzyme. The p r o p e r t i e s o f a n i s o l a t e d enzyme s y s t e m a r e l a r g e l y d e p e n d e n t o n t h e p u r i t y o f t h e enzyme and a l t h o u g h t h e g e n e r a l c h a r a c t e r i s t i c s may be e s t a b l i s h e d , t h e s p e c i f i c components o f t h e s y s t e m may n o t be d e t e r m i n e d w i t h a n y d e g r e e o f a c c u r a c y . The g l u c o s e o x i d i z i n g enzyme u n d e r s t u d y was n o t a p u r i f i e d enzyme p r e p a r a t i o n and t h e r e f o r e t h e r e s u l t s o b t a i n e d c a n o n l y be c o n s i d e r e d r e l a t i v e t o t h e s y s t e m . The g l u c o s e enzyme s y s t e m o f P . a e r u g i n o s a was f o u n d t o f u n c t i o n w h o l e and c o m p l e t e i n d r i e d c e l l p r e p a r a t i o n a n d was a b l e t o o x i d i z e g l u c o s e o v e r a w i d e r a n g e o f p H , ( 5 » 1 t o 8 . 0 ) . A l t h o u g h o x y g e n u p t a k e was o b s e r v e d f o r g l u c o s e o v e r t h i s e n t i r e r a n g e , g l u c o n i c a c i d c o u l d be i d e n t i f i e d a s t h e end p r o d u c t o f t h e r e a c t i o n o n l y a t p H 7 . 5 o r a b o v e . U n d e r a n a e r o b i c c o n d i t i o n s a t p H 7 . 5 t h e c e l l p r e p a r a t i o n was u n a b l e t o r e d u c e m e t h y l e n e b l u e i n t h e p r e s e n c e o f g l u c o s e . I t h a d b e e n f o u n d by o t h e r w o r k e r s ( 8 ) , ( 9 ) , ( 1 0 ) , ( 1 2 ) , ( 1 4 ) , ( 2 3 ) , t h a t m e t h y l e n e b l u e was c a p a b l e o f m e d i a t i n g t h e r e a c t i o n o f t h e i r s y s t e m when t e s t e d i n t h e p r e s e n c e o f s u b s t r a t e . P o t a s s i u m c y a n i d e v/as f o u n d t o i n h i b i t t h e r e a c t i o n i n c o n c e n t r a t i o n s - 39 -o f 10 r a n d 10~*^S i n d i c a t i n g t h e p o s s i b l e n e c e s s i t y o f c y t o c h r o m e o x i d a s e f o r t h e r e a c t i o n . On a d d i t i o n o f c y t o c h r o m e c t o t h e i n h i b i t e d f r a c t i o n and a l s o t o t h e d i a l y z e d enzyme no r e s t o r a t i o n o f a c t i v i t y was o b s e r v e d . A s t h e a d d i t i o n of c y t o c h r o m e c does n o t c o m p l e t e t h e c y t o c h r o m e c - c y t o c h r o m e o x i d a s e s y s t e m a g e n e r a l c o n c l u s i o n c a n n o t be made f r o m s t u d i e s o n c y a n i d e i n h i b i t i o n . H a r r i s o n ( 1 2 ) , H a w t h o r n e and H a r r i s o n ( 1 4 ) , and E i c h e l and W a i n i o (8 ) w i t h t h e i r enzyme p r e p a r a t i o n f r o m o x l i v e r f o u n d c y t o c h r o m e c - i n d o p h e n o l o x i d a s e c a t a l y z e d t h e o x i d a t i o n o f g l u c o s e t o g l u c o n i c and t h a t t h e s y s t e m was c y a n i d e s e n s i t i v e . The enzyme was f o u n d t o be h e a t l a b i l e , u n d e r g o i n g d e s t r u c t i o n a t t e m p e r a t u r e o f 55°^- f o r 5 m i n u t e s o r l e s s . A t 45° c. f o r p e r i o d o f 30 m i n u t e s a n d 3 7 ° C . f o r a p e r i o d o f 60 m i n u t e s t h e enzyme a c t i v i t y was f o u n d t o be impaired. O t h e r w o r k e r s ( $ ) , (9), ( l 0 ) , (27) h a v e p r e s e n t e d e v i d e n c e t h a t g l u c o s e o x i d a s e i s a f l a v o p r o t e i n o r h a v e s u g g e s t e d t h a t i t i s a f l a v i n b y f i n d i n g s t i m u l a t i o n i s p r o p o r t i o n t o added f l a v i n . The e f f e c t o f r i b o -f l a v i n on r e s t o r i n g t h e enzyme a c t i v i t y o f a d r i e d c e l l p r e p a r a t i o n f r o m P . a e r u g i n o s a when i t was p a r t i a l l y d e s t r o y e d b y l i g h t d i d n o t g i v e a n y i n d i c a t i o n a s t o t h e n a t u r e o f t h e p r o t e i n . H a d r i b o f l a v i n r e s t o r e d t h e s y s t e m t h e n t h e enzyme m i g h t h a v e b e e n c o n s i d e r e d a f l a v o p r o t e i n b y d e f i n i t i o n , d e s t r o y e d b y s t r o n g l i g h t a n d r e s t o r e d by added f l a v i n . A s f a r a s c a n be d e t e r m i n e d by a l i t e r a t u r e s u r v e y t h e g l u c o s e o x i d a s e o f P . a e r u g i n o s a i s t h e o n l y g l u c o s e o x i d i z i n g enzyme h a v i n g magnes ium a s i t s c o f a c t o r . O t h e r w o r k e r s h a v e r e c o r d e d c : d i p h o s p h o p y r i d i n e n u c l e o t i d e a s t h e coenzyme t o g l u c o s e o x i d a s e , h o w e v e r , i n t e s t i n g d i p h o s p h o p y r i d i n e n u c l e o t i d e o n t h e g l u c o s e o x i d a s e o f P . a e r u g i n o s a some i n h i b i t i o n was o b s e r v e d . A s i m i l a r f i n d i n g was o b t a i n e d when a d e n o s i n e - AO -t r i p h o s p h a t e was e m p l o y e d . F r o m t h e s e r e s u l t s i t i s c o n c l u d e d t h a t t h e g l u c o s e d i s s i m i l a t i n g enzyme o f P . a e r u g i n o s a i s n o t a d e h y d r o g e n a s e b u t a n o x i d a s e . I t i s h e a t l a b i l e , c y a n i d e s e n s i t i v e a n d r e q u i r e s magnes ium a s a c o f a c t o r . - 41 -SUMMARY 1. A d r i e d c e l l p r e p a r a t i o n o f P. a e r u g i n o s a was f o u n d t o m e d i a t e ± h e r e a c t i o n s g l u c o s e t o g l u c o n i c a c i d and g l u c o n i c a c i d t o 2 k e t o g l u c o n i c a c i d a t p H 7 . 0 . 2 . M e t h y l e n e b l u e , was f o u n d n o t t o f u n c t i o n as a h y d r o g e n a c c e p t o r f o r t h e a n a e r o b i c d i s s i m i l a t i o n o f g l u c o s e by a d r i e d c e l l p r e p a r a t i o n o f P. a e r u g i n o s a . 3. The p r o t e o l y t i c enzyme t r y p s i n was f o u n d t o d e s t r o y g l u c o s e o x i d a s e a c t i v i t y , w h i l e t a k a d i a s t a s e was w i t h o u t e f f e c t . A. S t r o n g l i g h t was f o u n d t o p a r t i a l l y i n a c t i v a t e g l u c o s e o x i d a s e . T h e a c t i v i t y o f t h e enzyme c o u l d n o t be r e s t o r e d by a d d i t i o n s o f r i b o f l v a i n . 5. G l u c o n i c a c i d was i d e n t i f i e d b y p a p e r c h r o m a t o g r a p h y a s a n end p r o d u c t o f t h e a e r o b i c o x i d a t i o n o f g l u c o s e a t pH7.5 by d r i e d c e l l p r e p a r a t i o n . 6. The enzyme a c t i o n was d e s t r o y e d by a t e m p e r a t u r e o f 55°G. f o r 5 m i n u t e s , a n d t h e r e was p a r t i a l d e s t r u c t i o n a t 43 °C. and 37 °C. a f t e r 60 m i n u t e s . 7 . The enzyme was i n h i b i t e d by c y a n i d e a n d a z i d e b u t was n o t e f f e c t e d b y m a l o n a t e , i o d o a c e t a t e o r a r s e n a t e . 8 . A p r o c e d u r e was e v o l w e d , u s i n g ammonium s u l p h a t e , f o r t h e s e p a r a t i o n o f t h e enzyme i n t o i t s two c o m p o n e n t s , apo and coenzyme . 9 . M a g n e s i u m was f o u n d t o f u n c t i o n a s a c o f a c t o r f o r g l u c o s e o x i d a s e and c o u l d be r e p l a c e d b y manganese . - 42 -BIBLIOGRAPHY 1. Barron, E.S.G. and Friedemann, T.E. Oxidation by organisms which do not ferment glucose. J. Biol. Chem. 137 : 593-610. 1941. 2. Campbell, J.J.R., Norris, F.C., and Norris, M.E. The intermediate metabolism of Pseudomonas aeruginosa. II. Limitations of simultaneous adaptation as applied to the identification of acetic acid, an intermediate in glucose oxidation. Can. J. Research, C, 27 : 165-171. 1949. 3. Campbell, J.J.R. and Norris, F.C. The intermediate metabolism of Pseudomonas aeruginosa. IV. The absence of an Embden-Meyerhof system as evidenced by phosphorus distribution. Can., J. Research, C, 28 : 203-212. 1950. 4. Cavallani, D., Frontali, N., and Toschi, G. Keto acid content of human blood and urine. Nature, 164 : 792-793* 1949. 5. Coulthard, C.E., Michaelis, R., Short, W.F., Sykes, G., Skrimshire, , G.E.H., Standfast, A.F.B., Brinkinshaw, J.H., and Raistrick, H. Notatin: an antibacterial glucose aerodehydrogenase from Penicillium notatum wealing. Nature, 150 : 634-635. 1942. 6. Coulthard, C.E., Michaelis, R., Short, W.F., Sykes, G., Skrimshire, G.E.H., Standfast, A.F.B., Brinkinshaw, J.H., and Raistrick, H. Notatin: an antibacterial glucose aerodehydrogenase from Penicillium notatum westling and Penicillium resticulosum Sp. nov. Biochem. J. 39 • 24-36. 1945. 7. Dickens, F. Oxidation of phosphohexonate and pentose phosphoric acids by yeast enzymes. Biochem. J. 32 : 1626-1653. 1938. 8 . Eichel, Bi and Wainio, W.W. D glucose dehydrogenase and its carrier systems. J. Biol. Chem. 175 : 155-168. 1948. 9 . Franke, W. and Deffner, M. Glucose Oxidase, II. Ann. 541 : 117-50. 1939. Cited in Chem. Abstracts, 34 : 3774 9. 1940. 10. Franke, W. and Lorenz, F. Glucose Oxidase, I. Ann. 532 : 1-28. 1937. Cited in Chem. Abstracts, 32 : 1731 7 . 1938. 11. Friedemann, T.E. and Haugen, G.E. The determination of keto acids in blood and urine. J. Biol. Chem. 147 : 415-441. 1943. 12. Harrison, D.C. Glucose dehydrogenase: A new oxidizing enzyme from animal tissue. Biochem. J. 25 : 1016-1027. 1931. 13. Harrison, D.C. Glucose dehydrogenase: of the enzyme and its co-enzyme. 1936. Preparation and some properties Biochem. J. 27 : 382-386. - 43 -14. Hawthorne, J.R. and Harrison, D.C. Cytochrome c as a carrier with the glucose dehydrogenase system. Biochem. J. 33 s 1573-1579. 1939. 15. Keilin, D. and Hartree, E.F. Prosthetic group of glucose oxidase (notatin). Nature, 157 : 801. 1946. 16. Krebs, H.A. The intermediary stages in biological oxidation of carbohydrate. Adv. in Enz. 3 : 191-252. 1943. , 17. Lipmann, F. Fermentation of phosphogluconic acid. Nature, 138 : 588. 1936. 18. Lipmann, F. Enzymatic synthesis of acetyl phosphate. J. Biol. Chem. 155 : 55-70. 1944. 19. Lockwood, L.B. and Stodola, F.H. Fermentation process for the production of c<ketoglutaric acid. U.S. Patent 2, 443, 919. 1948. 20. Lockwood, L.B., Tabenkin, B. and Ward, G.E. The production of gluconic acid and 2 ketogluconic acid from glucose by species of Pseudomonas and Phytomonas. J. Bact. 42 : 51-61. 1941. 21. Main, E.R. and Shina, L.E. The determination of hydrogen peroxide in bacterial cultures. J. Biol. Chem. 128 : 417-423. 1939. ' 22. Meyerhof, 0. Intermediate carbohydrate metabolism. Symp. on Resp. Enz. University of Wis. Press. 3-15. 1941. 23. Muller, D. Studies uber eines enzym glykoseoxydase. I. Biochem. Zeitshr. 199 : 136-170. 1928. Cited in Biol. Abstracts, 5 : 1319. 1931. 24. Muller, D. Glucose oxidase. Nafcurwiss. 28 : 516. 1940. Cited in Biol. Abstracts, 16 J 323. 1942. 25. Ney, P.W. A study of the intermediate metabolism of P. aeruginosa. -Thesis for M.S.A. degree. Dept. of Dairying. U.B.C. 1948. 26. Norris, F.C. and Campbell, J.J.R. The intermediate metabolism of Pseudomonas aeruginosa. III. The application of paper chromatography to the identification of gluconic and 2 keto-gluconic acids, intermediates in glucose oxidation. Can. J. Research, C, 27 : 253-261. 1949. 27. Ogston, F.J rand Green, D.E. The mechanism of the reaction of substrates with molecular oxygen. 1. Biochem. J. 29 : 1983-2004. 1935. I - AA -28. Osten, T.M. The production of resting cells of Pseudomonas aeruginosa suitable for the study of dehydrogenase activity. Thesis for B.S.A. degree. Dept. of Dairying. U.B.C. 1950. 29. Pickett, M.J. and Clifton, C.E. The effect of selective poisons on the utilization of glucose and intermediate compounds by microorganisms. J. Cell, and Comp. Physiol. 22 : 147-165. 1943. 30. Quastel, J.H. and Whetham, M.D. The equilibria existing between succinic fumaric and malic acids in the presence of resting bacteria. Biochem. J. 18 : 519-534-. 1924. 31. Robertson, W.V.B. The preparation of sodium pyruvate. Science, 96 : 93-94- 1942. 32. Stokes, F.C. and Campbell, J.J.R. The oxidation of glucose and gluconic acid by dried cells of Pseudomonas aeruginosa. Arch. Biochem. in Press. 1950. 33- Straub, F.B. Isolation and properties of a flavoprotein from heart muscle tissue. Biochem. J. 33 : 787-792. 1939. 34- • Sullivan, M. -Synthetic culture media and the biochemistry of bacterial pigments. J. Med. Research, 14 : 109-150. 1905. 35. Theorell, H. Biochem. Z. 278-263. 1935. Cited in Sumner, J.B. and Sommers, G.F. Chemistry and methods of enzymes. 279• 1947. 36. Warburg, 0. and Christian, W. Biochem. Z. 298 : 368. 1938. Cited in Sumner, J.B. and Sommers, G.F. Chemistry and methods of enzymes. 281. 1947. 

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