- Email: [email protected]
Breakdown of heptuloses in Escherichia coli
VOL. 17
(1955)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
595
Breakdown of heptuloses in Escherichia coli In view of the increasing interest in the role of sedoheptulose p h o s p h a t e in the m e t a b o l i s m o f glucose and pentose in animals 6, plants l, and m i c r o - o r g a n i s m sa, it appears worthwhile to r e p o r t s o m e e x p e r i m e n t s on tile degradation of various h e p t u l o s e s by t:~. coli, which have been carried o u t in the frame of a broader s t u d y to be reported elsewhere. The organism was cultivated on a mineral m e d i u m 3 containing 0.2 °i) glucose, D-xylose, D ribose or L-aral)inose, as well as on 0. 5 % },east e x t r a c t with and w i t h o u t 0.05 % glucose. Cells were harvested from 16 24 hour-old cultures, vigorously aerated at 37 ~ C. Oxidation was measured at 3 ° , by t h e conventional XVarburg techniques. The cells (25 mg of wet cells), suspended in o.05 fll phost)hate butter, p H 6.8, did not oxidize sedoheptulose, glucoheptulose, m a n n o h e p t u l o s e or s e d o h e p t u l o s a n m o n o h y d r a t e , nor did oxidation of sedoheptulose occur in the presence of glucose or ribo:se. I n a f u r t h e r series of experiments, the transfer of the terminal p h o s p h a t e group from adenosine t r i p h o s p h a t e (ATP) to heptuloses in cell-free extracts prepared by grinding the bacteria with a l u m i n a p o w d e r s was studied m a n o m e t r i c a l l y 4. Only the extract p r e p a r e d from cells g r o w n on yeast extract-glucose-medium, p h o s p h o r y h t t e d sedoheptulose to a considerable e x t e n t (Fig. i}. E x t r a c t s of cells grown on yeast e x t r a c t alone p h o s p h o r y l a t e d sedoheptulose slowly and only w h e n the c o n c e n t r a t i o n of the enzyme was increased five-fold (equivalent to tSO nag of wet cells). No other heptuloses were affected by the extracts. (Mannoheptulose was tested only with e x t r a c t s (7t" yeast-glucose grown cells.) Sedoheptulose is not only p h o s p h o r y l a t e d by tile ab!)ve extract, b u t is also oxidized {Fig. [). The inability of the corresponding intact cells to oxidize tile heptose coukt, therefore, be a t t r i b u t e d to a permeability barrier. T h a t the p h o s p h o r y l a t i o n p r o d u c t is p r o b a b l y f u r t h e r metabolized by extracts of P2. coli is indicated by the degradation of sedoheptulose-7-phosphate , which takes place in these extracts, as has previously been d e m o n s t r a t e d 2. Fig. 1. Oxidation and p h o s p h o r y l a t i o n of sedoheptulose b y extracts of E. coli grown on y e a s t - e x t r a c t glucose mcdimn. Oxidation (O - Q ) : tile flasks contained in the main c o m p a r t m e n t 0.9 ml e x t r a c t (equivalent to 18o mg wet cells); o.i ml (o.i mg) of triphosp h o p y r i d i n e nueleotide and o. i ml of MgC12. 6 H 2 0 (0.09 M). One side a r m carried 0. 3 mg of phenazine m e t h o s u l p h a t e in o.2 ml of tris buffer p H 7.o; in the second a r m : o.i m l o f A'I'P (sodium salt) (91~3I) and o.6 ml (9/till) substrate. The center well contained o.2 ml 2o % K O H . Final volume, 2.o ml; gas phase, air; t e m p e r a t u r e , 3 °o C. E n d o g e n o u s values subtracted. P h o s p h o r y l a t i o n : (A -l,) sedoheptulose, ( I . . . . m) endogenous; ( + -+) eluted c h r o m a t o g r a p h e d filter p a p e r treated as t h a t used in the p r e p a r a t i o n of sedoheptulose. Each flask contained in the main well, o.l 5 ml of bacterial extract (equivalent t o 3° mg wet cells); o. 4 ml of o.o8 M N a H C O a; o.~ ml of o. 4 J l K F ; o.1 ml of MgCI2.6H20 and o.2 ml of s u b s t r a t e (3/,:11). The side a r m received 9 !~.~I ATP in o. 4 ml of o.o2 31 N a H C O 3. Final volume, 2.o ml; gas phase, 75% N2 5°o COe; t e m p e r a t u r e , 3 ° : C.
I
~
I
I
I
I
200
I00 sO _z
160
60
t20 !N
40
eO 40
20 +
0
0 20
40
60
80
tO0
120
MINUTES
In this connection it m a y be interesting to record t h a t a red yeast, R h o d o l o r u l a m u c i l a g i n o s a , has been isolated in this l a b o r a t o r y from an exposed solution of m a n n o h e p t u l o s e and has been found to utilize this sugar as well as glucose. After this investigation had been completed, SATO el al. l° reported the isolation of a B a c i l l u s strain which can utilize sedoheptulose adaptively, and MOORE el al. ~ d e m o n s t r a t e d the utilization of sedoheptulose and the oxidation of heptoses b y various bacteria. Sedoheptulose was prepared b y hydrolysis of s e d o h e p t u l o s a n m o n o h y d r a t e in o.2 N HC1 for half an h o u r at IOO ~' C. I t was separated by descending c h r o m a t o g r a p h y ( W h a t m a n No. I), using w a t e r - s a t u r a t e d phenol as a solvent. The b a n d c o r r e s p o n d i n g to R F value 0.44 was cut out, washed with ether, and eluted with w a t e r b y the capillary descent procedure. Mannoheptulose was prepared, in collaboration with Dr. E. SIMON (\Veizmann I n s t i t u t e of Science) from avocado pears (Cnllinson variety) by the m e t h o d of LAFORGEL
590
SHORT (?OMMUNICATI()NS,
PREIdMINARV
NOTF.S
V()L. 1 7 ( i r i S h )
\Ve are g r e a t l y i n d e b t e d to Drs. B. L. t~ORECKER AND N. IX. RICHTMEYER, of t h e N a t i o n a l I n s t i t u t e of H e a l t h , B e t h e s d a , Md., for a g e n e r o u s g i f t of g l u c o h e p t u l o s e a n d s e d o h e p t u l o s a n m o n o h y d r a t e , a n d to P r o f e s s o r 51. :\SCItNER, (Jr t h e H e b r e w I ; n i v e r s i t y , J e r u s a l e m , for t h e i d e n t i t i ( a t i o n of t h e y e a s t . U. Z. IATTAUER The II'eicman~ [12sDl~le el Hcicm:e, lfet~uvo/h (Israel) B . E . V()I.CAN| .q~ ie~/:/ic l)el~arlme~zt, ~Ii~islry <7/ I)e/em'e (lsr~/el) E. l). I~ERGMANN R. S. BANDURSKI, C. M. GREINFR ANt) R. JAN(;,./. Hiv/. ('hem., 2o-' (1953) (Ji<). I". 1). FIERGMANN, U. Z. I.ITTAUFR AND t3. E. VOLCANL tJiochim, lJio]Sh),s..Icla, 13 (1954) ,SS. ~1 S. S. COHEN, .]. Hio/. Chem., 177 (1947) Oo 7. S. l >. COLOWICK AND H. M. KALCKAR, J. l~iol. Chem., 14S (t943) I l 7. "> M. GIBBS, l.. M. I>AFGE AND J. M. EAP.L, Hacterio/. Proc., (I954) ~ i. (; B. I.. HORECKER, I >. Z. SMVRXmTIS AND t l . KLENOW, .]. l?ioL Chem., -'o 5 (1953) 00~. 7 1,'. B. I.AFORG~'L .]. l~ioL Chem., 28 (~916) 5~7 . s 1t. MC)LWMN,./. (;e~. 31icrobioL, '- (I948) ~SS. !) \V. B. MOORF, A. C. BLACKWOOD AXD A. C. NEISH, ('atl../. 3licrobiol., t (]954) 19
R e c e i v e d M a y ~oth, ]055
On the prosthetic group of succinic dehydrogenase* '['he p r o b l e m of t h e p r o s t h e t i c g r o u p of s u c c i n i c d e h y d r o g e n a s e h a s b e e n t h e s u b j e c t ()1 mLa'h c ( m j e c t u r e , b u t a d e f i n i t i v e e x p e r i m e n t a l a p p r o a c h t o its s t u d y h a d t o a w a i t t h e i s o l a t i o n of t h e t / u r e e n z y m e . S i n c e t h e s u g g e s t i o n of .~kXELROD el al. l, b a s e d on u u t r i t i o n a I e x p e r i m e n t s t h a t Iluvi~ m a y b e t h e p r o s t h e t i c g r o u p of t h e e n z y m e , t h e p r e s e n c e of a flavin m o i e t y in t h e d e h y d r o g e n a s e h a s b e e n w i d e l y s p e c u l a t e d u p o n . O t h e r i n v e s t i g a t o r s f a v o r e d tile v i e w t h a t tile p r i m a r y d e h y d r ( ) g e n a s e is a h e m o p r o t e i n , p o s s i b l y i d e n t i c a l w i t h c y t o c h r o m e B. T h e a v a i l a l / i l i t y of e s s e n t i a l l y l l ( m l o g e n e o u s p r e p a r a t i o n s of t h e e n z y n l e e h a s o p e n e d tile w a y to a c r i t i c a l re e x a n l i n a t i o n of t h e prolJlem. T h e a u t h o r s h a v e r e p o r t e d t h a t t h e p r o s t h e t i c g r o u p of t h e d e h y d r o g e n a s e c o n t a i n s i r o n l i n t m~t in t h e form of heroin'2, :j. T h e t o t a l iron c o n t e n t e q u a l s t h e i n o r g a n i c i r o n w h i c h is l i b e r a t e d as Ire t 4 b y b o i l i n g or a c i d i f i c a t i o n . U l t r a c e n t r i f u g a l I y h o m o g e n e o u s p r e p a r a t i o n s of t h e d e h y d r o g e n a s e , p r e p a r e d from /~,esh m i t o c h o n d r i a l a c e t o n e p o w d e r s c o n t a i n l g a t o m of Fe- - p e r 0o,ooo to 05,ono g protein; similar preparations isolated from stored acetone powders (several mouths at l o ) contain l g a t o m F e - - p e r ~ t o , o o o t o I 3 o , o o o g. F r o m t h e p r e l i m i n a r y v a l u e of t h e m o l e c u l a r w e i g h t ( a b o u t ~4o,ooo, b a s e d on s e d i m e n t a t i o n r a t e a n d diffusion3), i t m a y be c o n c l u d e d t h a t t h e t w o t y p e s of p r e p a r a t i o n s c o n t a i n 2 a n d i g a t o m of F e ~; p e r mole, r e s p e c t i v e l y . S i n c e b y p h y s i c o - c h e m i c a l c r i t e r i a t h e t w o p r e p a r a t i o n s a p p e a r i d e n t i c a l a n d s i n c e t h e specific a c t i v i t y v a r i e s w i t h t h e iron c o n t e n t 2, it s e e m s t h a t b o t h iron a t o m s are n e e d e d for full a c t i v i t y a n d t h a t o n e of t h e t w o b e c o m e s r e a d i l y h d / i l i z e d a n d lost on s t o r a g e or p u r i f i c a t i o n . In c o n t r a s t , t h e i r o n in tile o n e - i r o n t y p e of p r e p a r a t i o n is so s t r o n g l y h e l d t h a t n e i t h e r d i a l y s i s a g a i n s t b u f f e r or F e + ~ - c h e l a t o r s n o r p a s s a g e t h r o u g h c a t i o n e x c h a n g e r s l i b e r a t e s it, a l t h o u g h it is s e t free on d e n a t u r a t i o n . Iron c o m p l e x o r s ( o - p h e n a n t h r o l i n e , 8 - o x y q u i n o l i n e , t h i o c y a n a t e , a,~F-bipyridyl, v e r s e n e , i r o n - s p e c i f i c v e r s e n e ) d o n o t i n h i b i t t h e e n z y m e b u t t h e a p o - p r o t e i n of c r y s t a l l i n e i l l - g l o b u l i n f r o m p l a s m a s t r o n g l y i n h i l ) i t s t h e d e h y d r o g e n a s e ; t h i s c a n be p r e v e n t e d a n d p a r t l y r e v e r s e d lay f e r r o u s iron. W h i l e o p h e n a n t h r o l i n e d o e s n o t i n h i b i t t h e e n z y m e , i t f o r m s a red c o m p l e x w i t h i t a n d tile r e s u l t i n g s p e c t r u m is s i m i l a r to b u t n o t i d e n t i c a l w i t h t h a t of f e r r o u s o - p h e n a n t h r o l i n a t e . T h e o - p h e n a n t h r o l i n e c o m p o u n d of s u c c i n i c d e h y d r o g e n a s e h a s full a c t i v i t y : it is l ) l e a c h e d b y s u c c i n a t e in t h e s a m e w a y as t h e free e n z y m e , a n d i t d o e s n o t d i s s o c i a t e on d i a l y s i s . T h e a b s o r p t i o n s p e c t r u m of s u c c i n i c d e h y d r o g e n a s e (Fig. l) is a t y p i c a l for a t l a w T p r o t e i n : t h e r e is no d e t i n i t e m a x i n m m in t h e r e g i o n of 46o or 375 In/*; t h e r e is a g r a d u a l l y i n c r e a s i n g a b s o r p t i o n b e l o w 4oo m/* as w e l l as a m e a s u r a b l e a b s o r p t i o n in t h e e n t i r e v i s i b l e r a n g e . T h e e n z y m e is p a r t l y b l e a c h e d b y h y d r o s u l f i t e a u d t h e r e s u l t i n g d i f f e r e n c e s p e c t r u m ( F i g . ) , insert) e x h i b i t s a m a x i n m m a t 46o m/,. S u c c i n a t e b l e a c h e s t h e e n z y m e less c o m p l e t e l y t h a n h y d r o s u l f i t e a n d t h i s r e d u c t i o n is inhilJited b y m a l o n a t e . T h e p r e p a r a t i o n s h o w n in Fig. i is t h e o n e - i r o n t y p e ; tile t w o - i r o n e n z y m e s h o w s a l m o s t 5 ° °'o m o r e c o l o r a t a n d t/elow 45o m/,, w h e r e a s a b o v e 52o In/i, w h e r e t h e iron m o i e t i e s ()l o t h e r f e r r o f l a v o p r o t e i n s a are t h o u g h t t o a b s o r b l i g h t , t h e t w o - i r o n e n z y m e s s h o w s t w i c e as m u c h color. * S u p p o r t e d b y a r e s e a r c h g r a n t from tile N a t i o n a l H e a r t I n s t i t u t e , I . S .
P u b l i c H e a l t h Service.
(1955)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
595
Breakdown of heptuloses in Escherichia coli In view of the increasing interest in the role of sedoheptulose p h o s p h a t e in the m e t a b o l i s m o f glucose and pentose in animals 6, plants l, and m i c r o - o r g a n i s m sa, it appears worthwhile to r e p o r t s o m e e x p e r i m e n t s on tile degradation of various h e p t u l o s e s by t:~. coli, which have been carried o u t in the frame of a broader s t u d y to be reported elsewhere. The organism was cultivated on a mineral m e d i u m 3 containing 0.2 °i) glucose, D-xylose, D ribose or L-aral)inose, as well as on 0. 5 % },east e x t r a c t with and w i t h o u t 0.05 % glucose. Cells were harvested from 16 24 hour-old cultures, vigorously aerated at 37 ~ C. Oxidation was measured at 3 ° , by t h e conventional XVarburg techniques. The cells (25 mg of wet cells), suspended in o.05 fll phost)hate butter, p H 6.8, did not oxidize sedoheptulose, glucoheptulose, m a n n o h e p t u l o s e or s e d o h e p t u l o s a n m o n o h y d r a t e , nor did oxidation of sedoheptulose occur in the presence of glucose or ribo:se. I n a f u r t h e r series of experiments, the transfer of the terminal p h o s p h a t e group from adenosine t r i p h o s p h a t e (ATP) to heptuloses in cell-free extracts prepared by grinding the bacteria with a l u m i n a p o w d e r s was studied m a n o m e t r i c a l l y 4. Only the extract p r e p a r e d from cells g r o w n on yeast extract-glucose-medium, p h o s p h o r y h t t e d sedoheptulose to a considerable e x t e n t (Fig. i}. E x t r a c t s of cells grown on yeast e x t r a c t alone p h o s p h o r y l a t e d sedoheptulose slowly and only w h e n the c o n c e n t r a t i o n of the enzyme was increased five-fold (equivalent to tSO nag of wet cells). No other heptuloses were affected by the extracts. (Mannoheptulose was tested only with e x t r a c t s (7t" yeast-glucose grown cells.) Sedoheptulose is not only p h o s p h o r y l a t e d by tile ab!)ve extract, b u t is also oxidized {Fig. [). The inability of the corresponding intact cells to oxidize tile heptose coukt, therefore, be a t t r i b u t e d to a permeability barrier. T h a t the p h o s p h o r y l a t i o n p r o d u c t is p r o b a b l y f u r t h e r metabolized by extracts of P2. coli is indicated by the degradation of sedoheptulose-7-phosphate , which takes place in these extracts, as has previously been d e m o n s t r a t e d 2. Fig. 1. Oxidation and p h o s p h o r y l a t i o n of sedoheptulose b y extracts of E. coli grown on y e a s t - e x t r a c t glucose mcdimn. Oxidation (O - Q ) : tile flasks contained in the main c o m p a r t m e n t 0.9 ml e x t r a c t (equivalent to 18o mg wet cells); o.i ml (o.i mg) of triphosp h o p y r i d i n e nueleotide and o. i ml of MgC12. 6 H 2 0 (0.09 M). One side a r m carried 0. 3 mg of phenazine m e t h o s u l p h a t e in o.2 ml of tris buffer p H 7.o; in the second a r m : o.i m l o f A'I'P (sodium salt) (91~3I) and o.6 ml (9/till) substrate. The center well contained o.2 ml 2o % K O H . Final volume, 2.o ml; gas phase, air; t e m p e r a t u r e , 3 °o C. E n d o g e n o u s values subtracted. P h o s p h o r y l a t i o n : (A -l,) sedoheptulose, ( I . . . . m) endogenous; ( + -+) eluted c h r o m a t o g r a p h e d filter p a p e r treated as t h a t used in the p r e p a r a t i o n of sedoheptulose. Each flask contained in the main well, o.l 5 ml of bacterial extract (equivalent t o 3° mg wet cells); o. 4 ml of o.o8 M N a H C O a; o.~ ml of o. 4 J l K F ; o.1 ml of MgCI2.6H20 and o.2 ml of s u b s t r a t e (3/,:11). The side a r m received 9 !~.~I ATP in o. 4 ml of o.o2 31 N a H C O 3. Final volume, 2.o ml; gas phase, 75% N2 5°o COe; t e m p e r a t u r e , 3 ° : C.
I
~
I
I
I
I
200
I00 sO _z
160
60
t20 !N
40
eO 40
20 +
0
0 20
40
60
80
tO0
120
MINUTES
In this connection it m a y be interesting to record t h a t a red yeast, R h o d o l o r u l a m u c i l a g i n o s a , has been isolated in this l a b o r a t o r y from an exposed solution of m a n n o h e p t u l o s e and has been found to utilize this sugar as well as glucose. After this investigation had been completed, SATO el al. l° reported the isolation of a B a c i l l u s strain which can utilize sedoheptulose adaptively, and MOORE el al. ~ d e m o n s t r a t e d the utilization of sedoheptulose and the oxidation of heptoses b y various bacteria. Sedoheptulose was prepared b y hydrolysis of s e d o h e p t u l o s a n m o n o h y d r a t e in o.2 N HC1 for half an h o u r at IOO ~' C. I t was separated by descending c h r o m a t o g r a p h y ( W h a t m a n No. I), using w a t e r - s a t u r a t e d phenol as a solvent. The b a n d c o r r e s p o n d i n g to R F value 0.44 was cut out, washed with ether, and eluted with w a t e r b y the capillary descent procedure. Mannoheptulose was prepared, in collaboration with Dr. E. SIMON (\Veizmann I n s t i t u t e of Science) from avocado pears (Cnllinson variety) by the m e t h o d of LAFORGEL
590
SHORT (?OMMUNICATI()NS,
PREIdMINARV
NOTF.S
V()L. 1 7 ( i r i S h )
\Ve are g r e a t l y i n d e b t e d to Drs. B. L. t~ORECKER AND N. IX. RICHTMEYER, of t h e N a t i o n a l I n s t i t u t e of H e a l t h , B e t h e s d a , Md., for a g e n e r o u s g i f t of g l u c o h e p t u l o s e a n d s e d o h e p t u l o s a n m o n o h y d r a t e , a n d to P r o f e s s o r 51. :\SCItNER, (Jr t h e H e b r e w I ; n i v e r s i t y , J e r u s a l e m , for t h e i d e n t i t i ( a t i o n of t h e y e a s t . U. Z. IATTAUER The II'eicman~ [12sDl~le el Hcicm:e, lfet~uvo/h (Israel) B . E . V()I.CAN| .q~ ie~/:/ic l)el~arlme~zt, ~Ii~islry <7/ I)e/em'e (lsr~/el) E. l). I~ERGMANN R. S. BANDURSKI, C. M. GREINFR ANt) R. JAN(;,./. Hiv/. ('hem., 2o-' (1953) (Ji<). I". 1). FIERGMANN, U. Z. I.ITTAUFR AND t3. E. VOLCANL tJiochim, lJio]Sh),s..Icla, 13 (1954) ,SS. ~1 S. S. COHEN, .]. Hio/. Chem., 177 (1947) Oo 7. S. l >. COLOWICK AND H. M. KALCKAR, J. l~iol. Chem., 14S (t943) I l 7. "> M. GIBBS, l.. M. I>AFGE AND J. M. EAP.L, Hacterio/. Proc., (I954) ~ i. (; B. I.. HORECKER, I >. Z. SMVRXmTIS AND t l . KLENOW, .]. l?ioL Chem., -'o 5 (1953) 00~. 7 1,'. B. I.AFORG~'L .]. l~ioL Chem., 28 (~916) 5~7 . s 1t. MC)LWMN,./. (;e~. 31icrobioL, '- (I948) ~SS. !) \V. B. MOORF, A. C. BLACKWOOD AXD A. C. NEISH, ('atl../. 3licrobiol., t (]954) 19
R e c e i v e d M a y ~oth, ]055
On the prosthetic group of succinic dehydrogenase* '['he p r o b l e m of t h e p r o s t h e t i c g r o u p of s u c c i n i c d e h y d r o g e n a s e h a s b e e n t h e s u b j e c t ()1 mLa'h c ( m j e c t u r e , b u t a d e f i n i t i v e e x p e r i m e n t a l a p p r o a c h t o its s t u d y h a d t o a w a i t t h e i s o l a t i o n of t h e t / u r e e n z y m e . S i n c e t h e s u g g e s t i o n of .~kXELROD el al. l, b a s e d on u u t r i t i o n a I e x p e r i m e n t s t h a t Iluvi~ m a y b e t h e p r o s t h e t i c g r o u p of t h e e n z y m e , t h e p r e s e n c e of a flavin m o i e t y in t h e d e h y d r o g e n a s e h a s b e e n w i d e l y s p e c u l a t e d u p o n . O t h e r i n v e s t i g a t o r s f a v o r e d tile v i e w t h a t tile p r i m a r y d e h y d r ( ) g e n a s e is a h e m o p r o t e i n , p o s s i b l y i d e n t i c a l w i t h c y t o c h r o m e B. T h e a v a i l a l / i l i t y of e s s e n t i a l l y l l ( m l o g e n e o u s p r e p a r a t i o n s of t h e e n z y n l e e h a s o p e n e d tile w a y to a c r i t i c a l re e x a n l i n a t i o n of t h e prolJlem. T h e a u t h o r s h a v e r e p o r t e d t h a t t h e p r o s t h e t i c g r o u p of t h e d e h y d r o g e n a s e c o n t a i n s i r o n l i n t m~t in t h e form of heroin'2, :j. T h e t o t a l iron c o n t e n t e q u a l s t h e i n o r g a n i c i r o n w h i c h is l i b e r a t e d as Ire t 4 b y b o i l i n g or a c i d i f i c a t i o n . U l t r a c e n t r i f u g a l I y h o m o g e n e o u s p r e p a r a t i o n s of t h e d e h y d r o g e n a s e , p r e p a r e d from /~,esh m i t o c h o n d r i a l a c e t o n e p o w d e r s c o n t a i n l g a t o m of Fe- - p e r 0o,ooo to 05,ono g protein; similar preparations isolated from stored acetone powders (several mouths at l o ) contain l g a t o m F e - - p e r ~ t o , o o o t o I 3 o , o o o g. F r o m t h e p r e l i m i n a r y v a l u e of t h e m o l e c u l a r w e i g h t ( a b o u t ~4o,ooo, b a s e d on s e d i m e n t a t i o n r a t e a n d diffusion3), i t m a y be c o n c l u d e d t h a t t h e t w o t y p e s of p r e p a r a t i o n s c o n t a i n 2 a n d i g a t o m of F e ~; p e r mole, r e s p e c t i v e l y . S i n c e b y p h y s i c o - c h e m i c a l c r i t e r i a t h e t w o p r e p a r a t i o n s a p p e a r i d e n t i c a l a n d s i n c e t h e specific a c t i v i t y v a r i e s w i t h t h e iron c o n t e n t 2, it s e e m s t h a t b o t h iron a t o m s are n e e d e d for full a c t i v i t y a n d t h a t o n e of t h e t w o b e c o m e s r e a d i l y h d / i l i z e d a n d lost on s t o r a g e or p u r i f i c a t i o n . In c o n t r a s t , t h e i r o n in tile o n e - i r o n t y p e of p r e p a r a t i o n is so s t r o n g l y h e l d t h a t n e i t h e r d i a l y s i s a g a i n s t b u f f e r or F e + ~ - c h e l a t o r s n o r p a s s a g e t h r o u g h c a t i o n e x c h a n g e r s l i b e r a t e s it, a l t h o u g h it is s e t free on d e n a t u r a t i o n . Iron c o m p l e x o r s ( o - p h e n a n t h r o l i n e , 8 - o x y q u i n o l i n e , t h i o c y a n a t e , a,~F-bipyridyl, v e r s e n e , i r o n - s p e c i f i c v e r s e n e ) d o n o t i n h i b i t t h e e n z y m e b u t t h e a p o - p r o t e i n of c r y s t a l l i n e i l l - g l o b u l i n f r o m p l a s m a s t r o n g l y i n h i l ) i t s t h e d e h y d r o g e n a s e ; t h i s c a n be p r e v e n t e d a n d p a r t l y r e v e r s e d lay f e r r o u s iron. W h i l e o p h e n a n t h r o l i n e d o e s n o t i n h i b i t t h e e n z y m e , i t f o r m s a red c o m p l e x w i t h i t a n d tile r e s u l t i n g s p e c t r u m is s i m i l a r to b u t n o t i d e n t i c a l w i t h t h a t of f e r r o u s o - p h e n a n t h r o l i n a t e . T h e o - p h e n a n t h r o l i n e c o m p o u n d of s u c c i n i c d e h y d r o g e n a s e h a s full a c t i v i t y : it is l ) l e a c h e d b y s u c c i n a t e in t h e s a m e w a y as t h e free e n z y m e , a n d i t d o e s n o t d i s s o c i a t e on d i a l y s i s . T h e a b s o r p t i o n s p e c t r u m of s u c c i n i c d e h y d r o g e n a s e (Fig. l) is a t y p i c a l for a t l a w T p r o t e i n : t h e r e is no d e t i n i t e m a x i n m m in t h e r e g i o n of 46o or 375 In/*; t h e r e is a g r a d u a l l y i n c r e a s i n g a b s o r p t i o n b e l o w 4oo m/* as w e l l as a m e a s u r a b l e a b s o r p t i o n in t h e e n t i r e v i s i b l e r a n g e . T h e e n z y m e is p a r t l y b l e a c h e d b y h y d r o s u l f i t e a u d t h e r e s u l t i n g d i f f e r e n c e s p e c t r u m ( F i g . ) , insert) e x h i b i t s a m a x i n m m a t 46o m/,. S u c c i n a t e b l e a c h e s t h e e n z y m e less c o m p l e t e l y t h a n h y d r o s u l f i t e a n d t h i s r e d u c t i o n is inhilJited b y m a l o n a t e . T h e p r e p a r a t i o n s h o w n in Fig. i is t h e o n e - i r o n t y p e ; tile t w o - i r o n e n z y m e s h o w s a l m o s t 5 ° °'o m o r e c o l o r a t a n d t/elow 45o m/,, w h e r e a s a b o v e 52o In/i, w h e r e t h e iron m o i e t i e s ()l o t h e r f e r r o f l a v o p r o t e i n s a are t h o u g h t t o a b s o r b l i g h t , t h e t w o - i r o n e n z y m e s s h o w s t w i c e as m u c h color. * S u p p o r t e d b y a r e s e a r c h g r a n t from tile N a t i o n a l H e a r t I n s t i t u t e , I . S .
P u b l i c H e a l t h Service.