- Email: [email protected]
Ethanolamine, a useful ligand for large scale purification of bacterial pyruvate dehydrogenase complex by affinity chromatography
Volume 85, number 1
FEBS LETTERS
J;mu~ry 1978
ETHANOLAMINE, A USEFUL LIGAND FOR L/~RGE SCALE PURIFICATION OF BACTERIAL PYRUVATE DEHYDROGENASE COMPLEX BY AFFINITY
CHROMATOGRAPHY
Jaap VISSER, Walter VAN DONGEN and Marijke STRATING Dept. o f Genetics, Agricultural University, Gen. Foulkesweg 53, Wageningen, The Netherlands Received 10 Novemtmr 1977
1. I n t r o d u c t i o n I n o u r l a b o r a t o r y we are i n t e r e s t e d in l~te bioc h e m i c a l genetic characterization o f pymvate
dehydr0genase complex mutants both in prokaryotes
matrices o f various length (Ca-Cs) based on both a l k y l a n d ~ - a m i n o a l k y l residues w i t h t h e p y r u v a t e d e h y d r o g e n a s e c o m p l e x o f E. coll. All these m a t r i c e s
demonstrated strong affinity for the multienzyme complex [13]. Introduction o f an ~-carboxyl moiety
(F,seherichia coli) a n d e u k a r y o t e s (Aspergillus nidulans). M o r e o v e r , e v o l u t i o n a r y aspects o f this m u l t i e n z y m e c o m p l e x h a v e o u r interest. A l t h o u g h various purificat i o n p r o c e d u r e s exist [ 1 - - 3 ] , o u r w o r k w o u l d b e n e f i t f r o m suitable a f f i n i t y c h r o m a t o g r a p h y s y s t e m s . A p a r t f r o m the a d v a n t a g e s f o r t h e p u r i f i c a t i o n o f m u t a n t e n z y m e s , s u c h s y s t e m s m a y p r o v i d e us w i t h a ref'med t o o l t o establish t h e n a t u r e o f c e r t a i n m u t a t i o n s . We have investigated t h e possibility o f a p p l y i n g t h e
leads t o c o m p l e t e loss o f a f f i n i t y . T h e h y d r o p h o b i c c h r o m a t o g r a p h y a p p r o a c h was t h e n n o t f u r t h e r c o n sidered as it t u r n e d o u t t o b e impossible to elute b o t h overall a c t i v i t y a n d l i p o a m i d e d e h y d r o g e n a s e a c t i v i t y u n d e r mild c o n d i t i o n s . In this p a p e r the e f f e c t o f i n t r o d u c i n g h y d r o x y l groups in t h e a l k y l chains will
vadous eofactors involved in the overall reaction.
2. Methods
T h i a m i n p y r o p h o s p h a t e h a s b e e n f o u n d t o be a useful ligand [4-] ; N A D + a n d A M P derivatives are still u n d e r investigation. Besides general lJgand aff'mity c h r o m a t o g r a p h y also the possibility t o use h y d r o p h o b i c c h r o m a t o g r a p h y in isolating the e n z y m e has b e e n e x p l o r e d . This a p p r o a c h s e e m s t h e r e f o r e w o r t h w h i l e as h y d r o p h o b i c inter-
actions contribute largely to the formation o f the stable and highly ordered structure of the pyruvate d e h y d r o g e n a s e c o m p l e x . D i h y d r o l i p o a m i d e transacetylase is k n o w n to p l a y a c e n t r a l role in this association [ 5 ] . I t was f o u n d b e f o r e t h a t l i p o a m i d e dehydrogenase, one of the constituent enzymes of t h e c o m p l e x , has i n d e e d afffmity f o r h y d r o c a r b o n c o a t e d S e p h a r o s e s [ 6 ] . We have t e s t e d h y d r o p h o b i c
be considered.
2.1. E n z y m e s o u r c e a n d isolation E. c e l l K I - 1 L R 8-13, a p y r u v a t e d e h y d r o g e n a s e c o m p l e x c o n s t i t u t i v e m u t a n t was g r o w n according to [ 2 ] . T h e c r u d e e x t r a c t was p r e p a r e d as described [2] using m i n o r m o d i f i c a t i o n s . T h e ceil~ ( 6 0 - 8 0 g b a c t e r i a l w e t w t ) were b r o k e n w i t h a n X-press ( L K B ) . T h e n 2 5 0 rnl e x t r a c t i o n b u f f e r was a d d e d . T h e 0.1 M p o t a s s i u m p h o s p h a t e e x t r a c t i o n b u f f e r , p H 7.0, cont a i n e d in a d d i t i o n to D N A a s e a n d R N A a s e ( 5 - - 1 0 g g / m l ) , 3 m M m a g n e s i u m chloride, 0.1 m M E D T A , 2 m M TPP, 2 m M d i t h i o t h r e i t o l or 5 m M fl-mercapl:oe t h a n o l a n d 50 I~M PMSF. T h e c e n t r i f u g a t i o n step was f o l l o w e d b y dialysis against 50 m M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.0, w h i c h c o n t a i n e d nUll
components mentioned above except for DNAaso, Abbreviations: TPP, thfamin pyrephos33hate; PMSF, phenylrnethylsulfon$,l fluoride; DTT, dithiothreitoI Elsevier/North-Holland B~'omedical Press
R N A a s e a n d TPP. O p e r a t i n g t e m p e r a t u r e in all steps was 4°C. Mg 2÷ is a d d e d since t h e e n z y m e p r e f e r e n t i 81
Volume 85, number 1
FEBS LETTERS
Fig.1. Affinity chromatography of pyruvate aelaydrogenase complex on ethanol--Sepharose 2B (column dimensions 2.5 X 30 cm) Crude extract was prepared from 60 g bacteria as described in section 2. Fractions of 15 ml were collected. A2so ( e - ~ ) and enzyme activity ( X - x ) are shown.
January ~_978
I
ii .4. 280
ally binds metal-TPP whereas EDTA is added in order to inactivate m e t a l requiring proteases.
2.2. Enzyme activity Pyruvate dehydrogenasx c o m p l e x overall activit.~z was measured according to [7] at 25°C. 2.3. l~latri?cpreparation Sepharose-2B was a c t i v a t e d with 1,4 b u t a n e d i o l d i ~ y c y d y l e t h e r according to [ 8 ] . The a c t i v a t e d m a t r i x was r e a c t e d w i t h 0 . ! M e t h a n o ! a m i n e in 0.2 M ~icarbonate b u f f e r at pH 9.5 during 24 h at r o o m temperature.
fract£ov,
2 . 4 . SDS--polyacrylamide gel electrophoresis
S t o c k s o l u t i o n s were m a d e according to [ 9 ] . R o u t i n e l y a s t a n d a r d gel percentage o f 10% was used with a 3% stacking gel. A c u r r e n t o f 3 m A / t u b c or 20 m A / s l a b was W e n - Staining o c c u r r e d with Coomassie brilliant blue R. Samples were dialyzed against 10 mM s o d i u m p h o s p h a t e b u f f e r pH 7.0 to remove p o t a s s i u m ions. W
3. Results a n d discussion When t r y i n g to prepare a t h i o c h r o m e - - S e p h a r o s e m a t r i x starting with e p o x y - a c t i v a t e d Sepharose we made an i n t e r e s t i n g discovery. Blocking the excess o f reactive groups was d o n e with e t h a n o l a m i n e as suggested b y Pharmacia Fine Chemica!s AB [ I 0 ] . In a c o n t r o l e x p e r i m e n t n o t h i o c h r o m e and o n l y ethanolamine was c o u p l e d . The l a t t e r material t u r n e d o u t to be a very effective affinity a d s o r b e n t for p y r u v a t e dehydrcg,~,ase from E. colt, and this i n t e r a c t i o n with the ligand is c o m p l e t e l y reversible. An increase in ionic strength e!utes the e n z y m e q u a n t i t a t i v e l y . CNBractivated Sepharose can also be used. F o r s t a b i l i t y reasons the e p o x y - d e r i v a t i z e d [ I 1] Sepharose was used. We have applied this principle for large scale purifications ( 6 0 - - 8 0 g bacterial wet wt). The initial 82
.~OO
//
W W
A
number
Volume 85, number 1
FEBS LETTERS
M
R
m
w
8
5
t2 Fig.2. SDS--polyaerylarnide disc gel eteetrophoresis patterns of different stages of pyruvate dehydrogenase complex purification. Protein, 10-50 #g, is applied/gel. (2.A) From left to right: pooled eluate (fraction !0--40); fraction 65, fraction 70; (2.B) Biogel chromatography in tl~e presence of TPP from left to right: starting matefiM; 3rd, 7th, lOth and 14tb_ fraction upon appearance of activity; (2.C) Bioge[ chromatography in the absence of TPP 3rd, 5th, 1lth and 13th fraction upon appearance of activity.
Ianuary ~.978
purification steps are ~ v e n in the methods. The a f ~Lc ' _, ~- 4 y matrix is used after the dialysis step (2.5 "< 35 cm column). Results are shown in fig. 1 whereas the electrophoretic analysis on SDS gels 'is ~wen in fig.2. The active fractions contain, besides the three characteristic c o m p o n e n t s and some low molecular weig~h~_ contaminants, breakdown products o f the transacetyt ase c o m p o n e n t (cf. [12]). The pur.'_fication in th~s step on the basis o f e n z y m e activity is approx. 80-fo!d whereas on the basis o f protein a 40-fold Purification is obtained. This discrepancy is due to the fhct that all our activity measurements, also those with the crude extract: have been performed aerobically. NADH oxidase activity in the crude extract is considerable, leading to an underestimation o f the activity present. The active fractions were precipitated with a m m o n i u m sulfate (65%). Further purification was obtained by Biogel A-50-M c h r o m a t o g r a p h y (column dimensions 2.5 X 90 cm) applying 6 0 - 1 0 0 mg protein at a time after dialysis against the equilibration buffer. The gel was equilibrated with 0.1 M potassium phosphate buffer pH 7.0, 5% ~ y c e r o l (v/v), 4 mM DTT, 3 mM _~_,-~gi~e~ium chloride and 2 mM 'I'PP. Elution was carried out in the same buffer. Under the conditions used, the intact complex separates well from those molecules contain•ng the characteristic transacetylase breakdown products and from low molecular weight contaminants. The presence o f TPP is crucial; -~,tkcrwise breakdown products are observed in all :'ractions as shown in fig.2. Separation between intT.-_:tcomple:~ molecules and those containing partially degraded - ol transacetylase on molecular wmgnt basis in the presence o f high TPP concentratiorts raises interesting questions like, is T P P j u s t protecting aga:tnst breakdown or does its effect involve reoequ~ibration o f the different subunits? We should like to point out that the use o f ethanolamine to block excess o f reactive groups m a y well lead to complications in other affinity c h r o m a t o g r a p h y systems. Particuiarly since in the absence o f Mg 2÷ more proteins are adsorbed from a crude extract, which is shown in fig.3. Moreover, the anaount o f protein adsorbed varies with the bacteria?~ species used. In the case o f Pseudomonas fluores.oens, e.g., a considerable a m o u n t o f adsorption takes place eve~_ in the presence o f Mg z÷. In small scale experiments we have als0 investigated the effect o f an increase in chain length, in all cases tested (C a - C s ) vhe results 83
Volume 85, number 1
FEBS LETTERS
A
Januz_ty 1978
B
Fig.3. Affinity chromatography of pyruvate dehydrogenase complex on ethanol--Sepharose and propanol--Sepharose ha the absence and presence of Mg~ in the elution buffer. Experiments were performed using 1.5 ml gel/column. Elufion of the enz~-me occurred by applying 0.3 M NaC! to the 50 mM phosphate elution buffer. SDS-gel electrophoresis patterns show from left to right: (3.A) Ethanol--Sepharose 2B. From left to centre, Mg~÷absent: initial eluate; active fraction. Frora centre to ~ h t , l~g~÷ (2.5 rm~,~)present: initial eluate, active fraction. (3.1]) Propanol--Sepharose 2B idem.
w e r e very similar. P y r u v a t e d e h y d r o g e n a s e c o m p l e x a c t i v i t y c o u l d be e l u t e d q u a n t i t a t i v e l y at t h e same m o l a r i t y ( 0 . 1 2 M) a p p l y i n g a p h o s p h a t e b u f f e r grad i e n t . H o w e v e r , as e x p e c t e d , a t h i g h e r chain l e n g t h s (C4, C~) t o t a l p r o t e i n was n o t r e c o v e r e d q u a n t i t a tively. M o r e o v e r , s o m e p r o t e i n s which h a r d l y a d s o r b on the C - - - S e p h a r o s e c o n t a m i n a t e t h e e n z y m e using higher c h a i n lengths. T h e p o t e n t i a l s o f these a d s o r b e n t s have n o t b e e n r i g o u r o u s l y t e s t e d t h o u g h , as n o g r a d i e n t e l u t i o n has b e e n a p p l i e d w h i c h raio~t i m p r o v e the results (cf. f i g . l ) . S o m e results are s h o w n in fig.3. We have t e s t e d w h e t h e r t h e a f f i n i t y c h r o m a t o g r a p h y s t e p is a p p l i c a b l e t o m u t a n t p y r u v a t e d e h y d r o genase c o m p l e x e n z y m e s f r o m / ~ : c o i l P u r i f i c a t i o n o f 4 d i f f e r e n t s t r u c t u r a l m u t a n t s tt us far leads to a similar o r even b e t t e r s t a t e o f p t r i t y as in t h e case o f wild t y p e in a similar stage o f t h ~ p r o c e d u r e . We have also a p p l i e d t h i s m e t h o d s u c c e s f j l l y t o o t h e r p r o 84
k a r y o t e s viz Azotobacger vit:e[andii, Bacillus sub ~t'lis, Bacillus polymyxa a n d Pseudomonas fluorescens. In s o m e cases t h o u g h , o t h e r i o n i c s t r e n g t h c o n d i t i o n s are r e q u i r e d f o r a d s o r p t i o n a n d d e s o r p f i o n ( t o be p u b l i s h e d else~.vhere). T h e m e t h o d p r e s e n t e d h e r e is c e r t a i n l y v e ~ valuable as it is m i l d a n d avoids b o t h protarrfine sulfate p r e c i p i t a t i o n a n d i s o e l e c t f i c f r a c t i o n a t i o n . We have c o m b i n e d a f f i n i t y c h r o m a t o ~raphy w i t h a m o l e c ular sieve p r o c e d u r e as t h e n e x t s t e p . I t m i g h t b e a d v a n t a g e o u s in o r d e r t o scale up t h e c o m p l e t e p u r i f i c a t i o n t o investigate whethe~r cancium p h o s p h a t e gel--~ce!!ulose c h r o m a t o ~ a p h y c a n be u s e d i n s t e a d o f t h e m o l e c u l a r sieving s t e p . We have n o t y e t d e t e r m i n e d w h e t h e r a p a r t i c u l a r i n d i v i d u a l c o m p o n e n t o f t h e e n z y m e c o m p l e n is r e q u i r e d o r t h a t bindLng depends o n t h e p r e s e n c e o f all c o m p o n e n t s . These p o i n t s will be e l u c i d a t e d b y
Volume 85, n u m b e r 1
FEBS LETTERS
dissociating the enzyme complex and by studying a p p r o p r i a t e m u t a n t e n z y m e ~ . We h a v e t e s t e d s o m e o t h e r m a t r i c e s t o establLsh t h e s t r u c t u r a l r e q u i r e m e n t s a I i g a n d h a s t o possess m o r d e r t o b e e f f e c t i v e . C o u pling ofl3-mercaptoethan01 and of glycerol both lead to products without affinity. The combination of a p o ~ t i v e l y c h a r g e d n i t r o g e n a n d a n a l c o h o l side chaLn seems to be necessary.
Aeknow|edgements T h e a u t h o r s w a n t t o t h a n k D r U. H e n n i n g for p r o v i d i n g us w i t h a c o n s t i t u t i v e p d c m u t a n t o f E s c h e r i c h i a coli. P a r t i c i p a t i o n o f D i c k v a n L i t h i n s o m e stages o f this w o r k is also a p p r e c i a t e d . Mr K. K n o o p skiHfuUy m a d e d r a w i n g s a n d p h o t o g r a p h s . D i s c u s s i o n s w i t h Drs C. 3. Bos a n d H. W. J. v a n d e n B r o e k are a p p r e c i a t e d .
Yanuary 1978
References [I] Reed, L. 3. and Mukherjee, B. B. (1969) in: Meth. EnzymoL 13, 5 5 - 6 1 . [2] Vogel, O., Beikirch, H., Miiller, H. and Henning, U. (1971) Eur. J. Biochem. 20, 1 6 9 - 1 7 8 . [3] Speckhard, D. C. and Frey, P. A. (1975) Biochem. Biophys. Res. Conlmun. 62. 614--620. [4] Visser, J., Strating, M. and Van Dongen, W. (1977) subm[tted. [5] Koike, M., Reed, L. J. and CarroU, W. R. (1960) J. Biol. Chem. 235, t924--1930. [6] Visser, J. and Strating, M. (1975) Biochim. 8iophys. Acta 384, 69--80. [7] Schwartz, E. R. and Reed, L. J. (1970) Biochemistry 9, 1434. [8] Sandberg, L. and Porath, J. (1974) J. Chromatog. 90, 87-98. [9] Laemmli, K. K. (1970) Nature 2 2 7 , 6 8 1 - 6 8 3 . [1G] Aff-mity chromatography, principles and methods (1974) p. 28, Pharmacia Fine Chemicals AB. [1t] Tesser, G. L, Fisch, H. U. and Schwijzer, .~. (1974) Helvetica Chimica Acta 57, 1 7 1 8 - 1 7 3 0 . [ i 2 ] P e r h a m R. N. and Thomas, J. O. (1971 ~EBS LETT. !5, 8--12. [ 13] Vissor, 3., Van Donjon, ~,V.and St_~ating, N[. unpublished.
85
FEBS LETTERS
J;mu~ry 1978
ETHANOLAMINE, A USEFUL LIGAND FOR L/~RGE SCALE PURIFICATION OF BACTERIAL PYRUVATE DEHYDROGENASE COMPLEX BY AFFINITY
CHROMATOGRAPHY
Jaap VISSER, Walter VAN DONGEN and Marijke STRATING Dept. o f Genetics, Agricultural University, Gen. Foulkesweg 53, Wageningen, The Netherlands Received 10 Novemtmr 1977
1. I n t r o d u c t i o n I n o u r l a b o r a t o r y we are i n t e r e s t e d in l~te bioc h e m i c a l genetic characterization o f pymvate
dehydr0genase complex mutants both in prokaryotes
matrices o f various length (Ca-Cs) based on both a l k y l a n d ~ - a m i n o a l k y l residues w i t h t h e p y r u v a t e d e h y d r o g e n a s e c o m p l e x o f E. coll. All these m a t r i c e s
demonstrated strong affinity for the multienzyme complex [13]. Introduction o f an ~-carboxyl moiety
(F,seherichia coli) a n d e u k a r y o t e s (Aspergillus nidulans). M o r e o v e r , e v o l u t i o n a r y aspects o f this m u l t i e n z y m e c o m p l e x h a v e o u r interest. A l t h o u g h various purificat i o n p r o c e d u r e s exist [ 1 - - 3 ] , o u r w o r k w o u l d b e n e f i t f r o m suitable a f f i n i t y c h r o m a t o g r a p h y s y s t e m s . A p a r t f r o m the a d v a n t a g e s f o r t h e p u r i f i c a t i o n o f m u t a n t e n z y m e s , s u c h s y s t e m s m a y p r o v i d e us w i t h a ref'med t o o l t o establish t h e n a t u r e o f c e r t a i n m u t a t i o n s . We have investigated t h e possibility o f a p p l y i n g t h e
leads t o c o m p l e t e loss o f a f f i n i t y . T h e h y d r o p h o b i c c h r o m a t o g r a p h y a p p r o a c h was t h e n n o t f u r t h e r c o n sidered as it t u r n e d o u t t o b e impossible to elute b o t h overall a c t i v i t y a n d l i p o a m i d e d e h y d r o g e n a s e a c t i v i t y u n d e r mild c o n d i t i o n s . In this p a p e r the e f f e c t o f i n t r o d u c i n g h y d r o x y l groups in t h e a l k y l chains will
vadous eofactors involved in the overall reaction.
2. Methods
T h i a m i n p y r o p h o s p h a t e h a s b e e n f o u n d t o be a useful ligand [4-] ; N A D + a n d A M P derivatives are still u n d e r investigation. Besides general lJgand aff'mity c h r o m a t o g r a p h y also the possibility t o use h y d r o p h o b i c c h r o m a t o g r a p h y in isolating the e n z y m e has b e e n e x p l o r e d . This a p p r o a c h s e e m s t h e r e f o r e w o r t h w h i l e as h y d r o p h o b i c inter-
actions contribute largely to the formation o f the stable and highly ordered structure of the pyruvate d e h y d r o g e n a s e c o m p l e x . D i h y d r o l i p o a m i d e transacetylase is k n o w n to p l a y a c e n t r a l role in this association [ 5 ] . I t was f o u n d b e f o r e t h a t l i p o a m i d e dehydrogenase, one of the constituent enzymes of t h e c o m p l e x , has i n d e e d afffmity f o r h y d r o c a r b o n c o a t e d S e p h a r o s e s [ 6 ] . We have t e s t e d h y d r o p h o b i c
be considered.
2.1. E n z y m e s o u r c e a n d isolation E. c e l l K I - 1 L R 8-13, a p y r u v a t e d e h y d r o g e n a s e c o m p l e x c o n s t i t u t i v e m u t a n t was g r o w n according to [ 2 ] . T h e c r u d e e x t r a c t was p r e p a r e d as described [2] using m i n o r m o d i f i c a t i o n s . T h e ceil~ ( 6 0 - 8 0 g b a c t e r i a l w e t w t ) were b r o k e n w i t h a n X-press ( L K B ) . T h e n 2 5 0 rnl e x t r a c t i o n b u f f e r was a d d e d . T h e 0.1 M p o t a s s i u m p h o s p h a t e e x t r a c t i o n b u f f e r , p H 7.0, cont a i n e d in a d d i t i o n to D N A a s e a n d R N A a s e ( 5 - - 1 0 g g / m l ) , 3 m M m a g n e s i u m chloride, 0.1 m M E D T A , 2 m M TPP, 2 m M d i t h i o t h r e i t o l or 5 m M fl-mercapl:oe t h a n o l a n d 50 I~M PMSF. T h e c e n t r i f u g a t i o n step was f o l l o w e d b y dialysis against 50 m M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.0, w h i c h c o n t a i n e d nUll
components mentioned above except for DNAaso, Abbreviations: TPP, thfamin pyrephos33hate; PMSF, phenylrnethylsulfon$,l fluoride; DTT, dithiothreitoI Elsevier/North-Holland B~'omedical Press
R N A a s e a n d TPP. O p e r a t i n g t e m p e r a t u r e in all steps was 4°C. Mg 2÷ is a d d e d since t h e e n z y m e p r e f e r e n t i 81
Volume 85, number 1
FEBS LETTERS
Fig.1. Affinity chromatography of pyruvate aelaydrogenase complex on ethanol--Sepharose 2B (column dimensions 2.5 X 30 cm) Crude extract was prepared from 60 g bacteria as described in section 2. Fractions of 15 ml were collected. A2so ( e - ~ ) and enzyme activity ( X - x ) are shown.
January ~_978
I
ii .4. 280
ally binds metal-TPP whereas EDTA is added in order to inactivate m e t a l requiring proteases.
2.2. Enzyme activity Pyruvate dehydrogenasx c o m p l e x overall activit.~z was measured according to [7] at 25°C. 2.3. l~latri?cpreparation Sepharose-2B was a c t i v a t e d with 1,4 b u t a n e d i o l d i ~ y c y d y l e t h e r according to [ 8 ] . The a c t i v a t e d m a t r i x was r e a c t e d w i t h 0 . ! M e t h a n o ! a m i n e in 0.2 M ~icarbonate b u f f e r at pH 9.5 during 24 h at r o o m temperature.
fract£ov,
2 . 4 . SDS--polyacrylamide gel electrophoresis
S t o c k s o l u t i o n s were m a d e according to [ 9 ] . R o u t i n e l y a s t a n d a r d gel percentage o f 10% was used with a 3% stacking gel. A c u r r e n t o f 3 m A / t u b c or 20 m A / s l a b was W e n - Staining o c c u r r e d with Coomassie brilliant blue R. Samples were dialyzed against 10 mM s o d i u m p h o s p h a t e b u f f e r pH 7.0 to remove p o t a s s i u m ions. W
3. Results a n d discussion When t r y i n g to prepare a t h i o c h r o m e - - S e p h a r o s e m a t r i x starting with e p o x y - a c t i v a t e d Sepharose we made an i n t e r e s t i n g discovery. Blocking the excess o f reactive groups was d o n e with e t h a n o l a m i n e as suggested b y Pharmacia Fine Chemica!s AB [ I 0 ] . In a c o n t r o l e x p e r i m e n t n o t h i o c h r o m e and o n l y ethanolamine was c o u p l e d . The l a t t e r material t u r n e d o u t to be a very effective affinity a d s o r b e n t for p y r u v a t e dehydrcg,~,ase from E. colt, and this i n t e r a c t i o n with the ligand is c o m p l e t e l y reversible. An increase in ionic strength e!utes the e n z y m e q u a n t i t a t i v e l y . CNBractivated Sepharose can also be used. F o r s t a b i l i t y reasons the e p o x y - d e r i v a t i z e d [ I 1] Sepharose was used. We have applied this principle for large scale purifications ( 6 0 - - 8 0 g bacterial wet wt). The initial 82
.~OO
//
W W
A
number
Volume 85, number 1
FEBS LETTERS
M
R
m
w
8
5
t2 Fig.2. SDS--polyaerylarnide disc gel eteetrophoresis patterns of different stages of pyruvate dehydrogenase complex purification. Protein, 10-50 #g, is applied/gel. (2.A) From left to right: pooled eluate (fraction !0--40); fraction 65, fraction 70; (2.B) Biogel chromatography in tl~e presence of TPP from left to right: starting matefiM; 3rd, 7th, lOth and 14tb_ fraction upon appearance of activity; (2.C) Bioge[ chromatography in the absence of TPP 3rd, 5th, 1lth and 13th fraction upon appearance of activity.
Ianuary ~.978
purification steps are ~ v e n in the methods. The a f ~Lc ' _, ~- 4 y matrix is used after the dialysis step (2.5 "< 35 cm column). Results are shown in fig. 1 whereas the electrophoretic analysis on SDS gels 'is ~wen in fig.2. The active fractions contain, besides the three characteristic c o m p o n e n t s and some low molecular weig~h~_ contaminants, breakdown products o f the transacetyt ase c o m p o n e n t (cf. [12]). The pur.'_fication in th~s step on the basis o f e n z y m e activity is approx. 80-fo!d whereas on the basis o f protein a 40-fold Purification is obtained. This discrepancy is due to the fhct that all our activity measurements, also those with the crude extract: have been performed aerobically. NADH oxidase activity in the crude extract is considerable, leading to an underestimation o f the activity present. The active fractions were precipitated with a m m o n i u m sulfate (65%). Further purification was obtained by Biogel A-50-M c h r o m a t o g r a p h y (column dimensions 2.5 X 90 cm) applying 6 0 - 1 0 0 mg protein at a time after dialysis against the equilibration buffer. The gel was equilibrated with 0.1 M potassium phosphate buffer pH 7.0, 5% ~ y c e r o l (v/v), 4 mM DTT, 3 mM _~_,-~gi~e~ium chloride and 2 mM 'I'PP. Elution was carried out in the same buffer. Under the conditions used, the intact complex separates well from those molecules contain•ng the characteristic transacetylase breakdown products and from low molecular weight contaminants. The presence o f TPP is crucial; -~,tkcrwise breakdown products are observed in all :'ractions as shown in fig.2. Separation between intT.-_:tcomple:~ molecules and those containing partially degraded - ol transacetylase on molecular wmgnt basis in the presence o f high TPP concentratiorts raises interesting questions like, is T P P j u s t protecting aga:tnst breakdown or does its effect involve reoequ~ibration o f the different subunits? We should like to point out that the use o f ethanolamine to block excess o f reactive groups m a y well lead to complications in other affinity c h r o m a t o g r a p h y systems. Particuiarly since in the absence o f Mg 2÷ more proteins are adsorbed from a crude extract, which is shown in fig.3. Moreover, the anaount o f protein adsorbed varies with the bacteria?~ species used. In the case o f Pseudomonas fluores.oens, e.g., a considerable a m o u n t o f adsorption takes place eve~_ in the presence o f Mg z÷. In small scale experiments we have als0 investigated the effect o f an increase in chain length, in all cases tested (C a - C s ) vhe results 83
Volume 85, number 1
FEBS LETTERS
A
Januz_ty 1978
B
Fig.3. Affinity chromatography of pyruvate dehydrogenase complex on ethanol--Sepharose and propanol--Sepharose ha the absence and presence of Mg~ in the elution buffer. Experiments were performed using 1.5 ml gel/column. Elufion of the enz~-me occurred by applying 0.3 M NaC! to the 50 mM phosphate elution buffer. SDS-gel electrophoresis patterns show from left to right: (3.A) Ethanol--Sepharose 2B. From left to centre, Mg~÷absent: initial eluate; active fraction. Frora centre to ~ h t , l~g~÷ (2.5 rm~,~)present: initial eluate, active fraction. (3.1]) Propanol--Sepharose 2B idem.
w e r e very similar. P y r u v a t e d e h y d r o g e n a s e c o m p l e x a c t i v i t y c o u l d be e l u t e d q u a n t i t a t i v e l y at t h e same m o l a r i t y ( 0 . 1 2 M) a p p l y i n g a p h o s p h a t e b u f f e r grad i e n t . H o w e v e r , as e x p e c t e d , a t h i g h e r chain l e n g t h s (C4, C~) t o t a l p r o t e i n was n o t r e c o v e r e d q u a n t i t a tively. M o r e o v e r , s o m e p r o t e i n s which h a r d l y a d s o r b on the C - - - S e p h a r o s e c o n t a m i n a t e t h e e n z y m e using higher c h a i n lengths. T h e p o t e n t i a l s o f these a d s o r b e n t s have n o t b e e n r i g o u r o u s l y t e s t e d t h o u g h , as n o g r a d i e n t e l u t i o n has b e e n a p p l i e d w h i c h raio~t i m p r o v e the results (cf. f i g . l ) . S o m e results are s h o w n in fig.3. We have t e s t e d w h e t h e r t h e a f f i n i t y c h r o m a t o g r a p h y s t e p is a p p l i c a b l e t o m u t a n t p y r u v a t e d e h y d r o genase c o m p l e x e n z y m e s f r o m / ~ : c o i l P u r i f i c a t i o n o f 4 d i f f e r e n t s t r u c t u r a l m u t a n t s tt us far leads to a similar o r even b e t t e r s t a t e o f p t r i t y as in t h e case o f wild t y p e in a similar stage o f t h ~ p r o c e d u r e . We have also a p p l i e d t h i s m e t h o d s u c c e s f j l l y t o o t h e r p r o 84
k a r y o t e s viz Azotobacger vit:e[andii, Bacillus sub ~t'lis, Bacillus polymyxa a n d Pseudomonas fluorescens. In s o m e cases t h o u g h , o t h e r i o n i c s t r e n g t h c o n d i t i o n s are r e q u i r e d f o r a d s o r p t i o n a n d d e s o r p f i o n ( t o be p u b l i s h e d else~.vhere). T h e m e t h o d p r e s e n t e d h e r e is c e r t a i n l y v e ~ valuable as it is m i l d a n d avoids b o t h protarrfine sulfate p r e c i p i t a t i o n a n d i s o e l e c t f i c f r a c t i o n a t i o n . We have c o m b i n e d a f f i n i t y c h r o m a t o ~raphy w i t h a m o l e c ular sieve p r o c e d u r e as t h e n e x t s t e p . I t m i g h t b e a d v a n t a g e o u s in o r d e r t o scale up t h e c o m p l e t e p u r i f i c a t i o n t o investigate whethe~r cancium p h o s p h a t e gel--~ce!!ulose c h r o m a t o ~ a p h y c a n be u s e d i n s t e a d o f t h e m o l e c u l a r sieving s t e p . We have n o t y e t d e t e r m i n e d w h e t h e r a p a r t i c u l a r i n d i v i d u a l c o m p o n e n t o f t h e e n z y m e c o m p l e n is r e q u i r e d o r t h a t bindLng depends o n t h e p r e s e n c e o f all c o m p o n e n t s . These p o i n t s will be e l u c i d a t e d b y
Volume 85, n u m b e r 1
FEBS LETTERS
dissociating the enzyme complex and by studying a p p r o p r i a t e m u t a n t e n z y m e ~ . We h a v e t e s t e d s o m e o t h e r m a t r i c e s t o establLsh t h e s t r u c t u r a l r e q u i r e m e n t s a I i g a n d h a s t o possess m o r d e r t o b e e f f e c t i v e . C o u pling ofl3-mercaptoethan01 and of glycerol both lead to products without affinity. The combination of a p o ~ t i v e l y c h a r g e d n i t r o g e n a n d a n a l c o h o l side chaLn seems to be necessary.
Aeknow|edgements T h e a u t h o r s w a n t t o t h a n k D r U. H e n n i n g for p r o v i d i n g us w i t h a c o n s t i t u t i v e p d c m u t a n t o f E s c h e r i c h i a coli. P a r t i c i p a t i o n o f D i c k v a n L i t h i n s o m e stages o f this w o r k is also a p p r e c i a t e d . Mr K. K n o o p skiHfuUy m a d e d r a w i n g s a n d p h o t o g r a p h s . D i s c u s s i o n s w i t h Drs C. 3. Bos a n d H. W. J. v a n d e n B r o e k are a p p r e c i a t e d .
Yanuary 1978
References [I] Reed, L. 3. and Mukherjee, B. B. (1969) in: Meth. EnzymoL 13, 5 5 - 6 1 . [2] Vogel, O., Beikirch, H., Miiller, H. and Henning, U. (1971) Eur. J. Biochem. 20, 1 6 9 - 1 7 8 . [3] Speckhard, D. C. and Frey, P. A. (1975) Biochem. Biophys. Res. Conlmun. 62. 614--620. [4] Visser, J., Strating, M. and Van Dongen, W. (1977) subm[tted. [5] Koike, M., Reed, L. J. and CarroU, W. R. (1960) J. Biol. Chem. 235, t924--1930. [6] Visser, J. and Strating, M. (1975) Biochim. 8iophys. Acta 384, 69--80. [7] Schwartz, E. R. and Reed, L. J. (1970) Biochemistry 9, 1434. [8] Sandberg, L. and Porath, J. (1974) J. Chromatog. 90, 87-98. [9] Laemmli, K. K. (1970) Nature 2 2 7 , 6 8 1 - 6 8 3 . [1G] Aff-mity chromatography, principles and methods (1974) p. 28, Pharmacia Fine Chemicals AB. [1t] Tesser, G. L, Fisch, H. U. and Schwijzer, .~. (1974) Helvetica Chimica Acta 57, 1 7 1 8 - 1 7 3 0 . [ i 2 ] P e r h a m R. N. and Thomas, J. O. (1971 ~EBS LETT. !5, 8--12. [ 13] Vissor, 3., Van Donjon, ~,V.and St_~ating, N[. unpublished.
85