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Purification of Acetate Kinase by Affinity Chromatography a
James R. Swartz , Gary W. Pace A. Solomon Archer
a c
a b
, Barry
a
, Clark K. Colton & Michael C.
a
a
Department of Nutrition and Food Science (JRS, GWP, MCA) and Department of Chemical Engineering (BAS, CKC) , Massachusetts Institute of Technology , Cambridge, Massachusetts, 02139 b
Tate and Lyle Ltd. , Reading, England
c
Amicon Corp. , Lexington, MA. Published online: 05 Dec 2006.
To cite this article: James R. Swartz , Gary W. Pace , Barry A. Solomon , Clark K. Colton & Michael C. Archer (1978) Purification of Acetate Kinase by Affinity Chromatography, Preparative Biochemistry, 8:6, 479-502, DOI: 10.1080/00327487808061665 To link to this article: http://dx.doi.org/10.1080/00327487808061665
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PREPARATIVE BIOCHEMISTRY, 8(6), 479-502 (1978)
PURIFICATION O F ACETATE KINASE BY AFFINITY CHROMATOGRAPHY
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James R. S w a r t z , Gary W. C l a r k K.
Pace,'
B a r r y A. S o l o m o n I 2
C o l t o n a n d M i c h a e l C. A r c h e r 3
Department o f N u t r i t i o n and Food S c i e n c e (JRS, GWP, MCA) and Department o f Chemical E n g i n e e r i n g (BAS, C K C ) , Mas sac hu s et t s I n s ti t u t e o f Techno 1og y , Cambridge, M a s s a c h u s e t t s
02 1 3 9
ABSTRACT A one-step procedure u s i n g a f f i n i t y chromatography
h a s b e e n shown t o p u r i f y t o a p p a r e n t homogeneity ace-
t a t e k i n a s e from a c o m m e r c i a l l y a v a i l a b l e p r e p a r a t i o n and t o p a r t i a l l y p u r i f y t h e enzyme from a c r u d e , c e l l f r e e extract.
S i n c e t h e g e l ' s c a p a c i t y f o r enzyme ad-
s o r p t i o n i s c o n t r o l l e d by t h e thermodynamics o f l i g a n d enzyme i n t e r a c t i o n , m a x i m i z a t i o n o f t h e a d s o r p t i o n isotherm w a s attempted.
Enzyme a d s o r p t i o n d e c r e a s e d
l o g a r i t h m i c a l l y w i t h i n c r e a s i n g i o n i c s t r e n g t h b u t inc r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f MgC12.
These
c o m p e t i n g e f f e c t s c a u s e d t h e . n e t a d s o r p t i o n of enzyme
479 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
480
SWART2 ET AL.
t o i n c r e a s e t o a maximum and t h e n t o d e c r e a s e a s t h e MgC12 c o n c e n t r a t i o n was r a i s e d .
The r e s u l t s allow a
s i g n i f i c a n t improvement i n a f f i n i t y column performance and have i m p o r t a n t i m p l i c a t i o n s f o r s c a l e - u p p r o c e d u r e s .
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INTRODUCTION
F i o s y n t h e s i s of many chemicals i n v o l v e s use o f t h e c o f a c t o r a d e n o s i n e t r i p h o s p h a t e (ATP) as a n e n e r g y source.
The economic a p p l i c a t i o n o f enzymes as c a t a -
lysts for industrial-scale synthesis therefore requires t h e development o f a n i n e x p e n s i v e p r o c e s s € o r r e g e n e r a t i o n o f ATP from a d e n o s i n e d i p h o s p h a t e (ADP) o r m n o phosphate (AMP).
The r e l a t i v e merits of v a r i o u s r o u t e s
t o ATP r e g e n e r a t i o n have been e v a l u a t e d 4 ; t h e s e i n v o l v e chemical s y n t h e s i s , whole c e l l s o r o r g a n e l l e s , or c e l l f r e e enzymes. f e r a s e , E.C.
Acetate k i n a s e (ATP :acetate phosphotrans2.7.2.1)
r e g e n e r a t i o n scheme.
i s a key enzyme i n a promising Procedures r e p o r t e d f o r i s o l a t i o n
and p u r i f i c a t i o n o f acetate k i n a s e i n v o l v e a number of s t e p s , i n c l u d i n g p r e c i p i t a t i o n by a c e t o n e and ammonium s u l f a t e , and g e l - and ion-exchange chromatography.
B
I n view of t h e p o t e n t i a l u s e o f acetate k i n a s e f o r ATP r e g e n e r a t i o n , a s i m p l e r and more e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e seems d e s i r a b l e . I n t h e p r e s e n t p a p e r , we d e s c r i b e a s i n g l e - s t e p a f f i n i t y chromatography p r o c e d u r e u s i n g AMP d e r i v a t i v e s of a g a r o s e f o r t h e 38-fold p u r i f i c a t i o n of acetate
4 81
ACETATE KINASE
k i n a s e from a c r u d e , c e l l - f r e e e x t r a c t o f E s c h e r i c h i a
coli.
The r e s u l t i n g p a r t i a l l y p u r i f i e d p r e p a r a t i o n i s
s u i t a b l e f o r d i r e c t u s e i n an ATP-regeneration reactor because it c o n t a i n s n o p r o t e a s e , p h o s p h a t a s e , ATPase o r
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adenylate kinase a c t i v i t i e s .
We a l s o d e s c r i b e t h e p u r i -
f i c a t i o n t o a p p a r e n t homogeneity of a commercially available a c e t a t e kinase preparation. S i n c e t h i s method may f i n d u s e i n i n d u s t r i a l - s c a l e enzyme p r o d u c t i o n , w e have i n v e s t i g a t e d c e r t a i n a s p e c t s of t h e a d s o r p t i o n p r o c e s s t h a t i n f l u e n c e s e p a r a t i o n performance.
The c a p a c i t y o f t h e a f f i n i t y s u p p o r t was
q u a n t i t a t e d i n terms o f t h e p r o d u c t of t h e e f f e c t i v e l i g a n d c o n c e n t r a t i o n and t h e thermodynamic e q u i l i b r i u m c o n s t a n t f o r enzyme-ligand
interaction.
Purification
performance was s i g n i f i c a n t l y enhanced by t h e i d e n t i f ic a t i o n and o p t i m i z a t i o n of p a r a m e t e r s t h a t i n f l u e n c e t h e e q u i l i b r i u m c o n s t a n t f o r enzyme a d s o r p t i o n .
This
i n f o r m a t i o n w i l l be o f v a l u e n o t o n l y f o r i n c r e a s i n g p r o c e s s scale, b u t f o r u n d e r s t a n d i n g t h e thermodynamics and mechanism of a f f i n i t y a d s o r p t i o n methods i n g e n e r a l . MATERIALS AND METHODS
-E. c o l i acetate k i n a s e , y e a s t hexokinase, and g l u cose-6-phosphate
dehydrogenase were o b t a i n e d a s suspen-
s i o n s i n 3 . 2 M ammonium s u l f a t e from Sigma ( S t . L o u i s ) . D i t h i o t h r e i t o l ( DTT) , ADP, AMP, nicotinamide-adenine d i n u c l e o t i d e phosphate (NADP), and a c e t y l phosphate
w e r e a l s o o b t a i n e d from Sigma.
S W T Z ET AL.
482
The a f f i n i t y chromatography g e l s u p p o r t s used i n t h i s s t u d y were o b t a i n e d i n preswollen form from P.L. Biochemicals, I n c .
(Milwaukee), and are d e s i g n a t e d by
t h e manufacturer a s agarose-hexane adenosine 5 I-phos-
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p h a t e (AGAMP) t y p e s 2 , 3 , and 4 .
They c o n s i s t o f AMP
a t t a c h e d t o a n a q a r o s e s u p p o r t by a six-carbon
spacer
through e i t h e r t h e amino group ( t y p e 2 1 , t h e C 8 of t h e adenine moiety ( t y p e 3 ) , o r t h e hydroxyl groups on t h e ribose ring (type 4 ) .
The c o n c e n t r a t i o n o f t h e AMP li-
qand, a s r e p o r t e d by t h e manufacturer, i s 5.8 pmol/ml packed w e t g e l ( t y p e 3 ) . P r e p a r a t i o n of Crude Acetate Kinase
-E. c o l i
(ATCC 9637-1,
w i l d t y p e ) w a s grown anaero-
b i c a l l y a t 37OC on a medium c o n t a i n i n g ( p e r l i t e r ) : 4 q glucose, KH2P04,
1.4
0.5
g ( N H 4 I 2 S O 4 , 0.5
g MgS04, 0.9 g
g N a 2 H P 0 4 , 0.11 mg C a C 1 2 ,
0.22 mg CuSO4,
0.15 mg FeS04, 0 . 2 1 mg Na2Mo04, 0.13 mg CoC12, 0.60 mg MnS04, and 0.81 mg ZnS04.
A crude c e l l - f r e e e x t r a c t
c o n t a i n i n g acetate k i n a s e a c t i v i t y w a s prepared from t h e h a r v e s t e d E. c o l i by r e s u s p e n s i o n of t h e c e l l s i n 0 . 1 M phosphate b u f f e r (pH 7 . 4 1 ,
d i s r u p t i o n i n a Braun
r o t a r y homogenizer, and c e n t r i f u g a t i o n a t 1 5 , 0 0 0 x 9: f o r 1 5 minutes t o remove c e l l d e b r i s .
The s u p e r n a t a n t
c o n t a i n e d approximately 1 5 U acetate k i n a s e p e r m g protein.
This crude e x t r a c t w a s s t o r e d i n l i q u i d n i t r o g e n .
To p r e v e n t l o s s of a c t i v i t y by o x i d a t i o n of s u l f h y d r y l g r o u p s r 7 1 . 0 mM DTT was added t o a l l enzyme s o l u t i o n s .
483
ACETATE KINASE
A d d i t i o n o f DTT t o t h e p a r t i a l l y p u r i f i e d a c e t a t e kin-
ase from Sigma i n c r e a s e d a c t i v i t y from 1 7 0 U p e r mg prot e i n , a s r e p o r t e d by Sigma, o r 2 2 0
u
p e r mg p r o t e i n , a s
measured by o u r a s s a y , t o 930 U p e r mg p r o t e i n .
Since
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t h e commercial enzyme was o b t a i n e d a s a s u s p e n s i o n i n c o n c e n t r a t e d (NH4)2SOqr it w a s c o n v e n i e n t t o a d j u s t a l l enzyme s o l u t i o n s t o a uniform c o n c e n t r a t i o n ( 2 6 mM) of ( N H 4 ) 2so4.
One u n i t of acetate k i n a s e was d e f i n e d as t h e amount o f enzyme t h a t c a t a l y z e s t h e p r o d u c t i o n of one pmol A T P p e r minute when added t o t h e f o l l o w i n g a s s a y solution:
5 mM ADP,
5 mM a c e t y l p h o s p h a t e , 1 0 mM glu-
c o s e , 0.3 mM NADP, 1 0 mM MgC12, 5 U/ml hexokinase, and 2 U/ml
glucose-6-phosphate
b u f f e r a t pH 1 . 4 and 3OOC.
dehydrogenase i n 0.1 M T r i s Formation of NADPH w a s
monitored c o n t i n u o u s l y by s p e c t r o p h o t o m e t r y a t 34 0 nm. Adenylate k i n a s e a c t i v i t y w a s determined by t h e same c o n t i n u o u s a s s a y e x c e p t t h a t a c e t y l phosphate w a s n o t included i n t h e assay solution.
When b o t h enzymes were
p r e s e n t , t h e measured acetate k i n a s e a c t i v i t y w a s c o r r e c t e d by s u b t r a c t i n g from it one h a l f of t h e adenyla t e k i n a s e a c t i v i t y , which w a s less t h a n 1 0 % o f t h e
acetate k i n a s e a c t i v i t y i n t h e c r u d e e x t r a c t and w a s n e g l i g i b l e i n t h e commercial p r e p a r a t i o n . p r o t e i n was monitored e i t h e r by s p e c t r o p h o t o m e t r y a t 280 nm o r by t h e Lowry p r o c e d u r e 8 a f t e r d i a l y s i s t o remove t h e i n t e r f e r i n g DTT.
SWART2 ET AL.
484
D i s c g e l e l e c t r o p h o r e s i s w a s performed according
t o t h e procedure of G a b r i e l g f o r an a n i o n i c enzyme sample (system I , 7.5% acrylamide)
.
I n characterizing
t h e enyzme s o l u t i o n p u r i f i e d from E . c o l i , phosphatase
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a c t i v i t y w a s determined by t h e method of F i s k e and Subbarow.lo
P r o t e a s e a c t i v i t y w a s determined by incub-
a t i n g t h e s o l u t i o n i n c o n t a c t w i t h c a s e i n a t 4OoC f o r one hour.
Any change i n t h e l e v e l o f n o n p r e c i p i t a b l e
p r o t e i n s w a s t h e n a s s a y e d by t h e Lowry procedure.0 Column P u r i f i c a t i o n A l l p u r i f i c a t i o n s were carried o u t a t pH 7.4
and
room temperature, i n a mode p r o p e r l y termed a f f i n i t y o r b i o s p e c i f i c adsorption.
A solution containing the
enzyme t o be p u r i f i e d w a s a p p l i e d t o a column ( 9 mm x 6 c m ) c o n t a i n i n g 2-4
m l o f a f f i n i t y gel.
When t h e
column was s a t u r a t e d (denoted by t h e c o n c e n t r a t i o n of enzyme i n t h e e x i t stream exceeding 90% of t h a t i n t h e i n l e t stream), enzyme a d d i t i o n was terminated.
The
column w a s t h e n washed w i t h 8 m l o f low-ionic s t r e n g t h s o l u t i o n c o n t a i n i n g o n l y 1 mM DTT and a c o n c e n t r a t i o n o f MgC12 e q u a l t o t h a t i n t h e enzyme s o l u t i o n a p p l i e d t o t h e column.
The acetate k i n a s e w a s e l u t e d w i t h a
s o l u t i o n of 1 0 mM ADP and 1 mM DTT, c o n t a i n i n g t h e same c o n c e n t r a t i o n of MgC12.
Sample a l i q u o t s (1-4 m l ) o f
e f f l u e n t were c o l l e c t e d and assayed.
A f t e r each use,
t h e g e l w a s washed w i t h 1 M N a C l t o r e m v e n o n s p e c i f i -
485
ACETATE KINASE
c a l l y bound p r o t e i n and s t o r e d a t 5OC i n 0.02% sodium a z i d e s o l u t i o n u n t i l reuse. Equilibrium Binding S t u d i e s The c a p a c i t y o f t h e g e l f o r enzyme-ligand i n t e r -
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a c t i o n was e v a l u a t e d from t h e r e s u l t s of column p u r i f i c a t i o n experiments and a l s o from e x p e r i m e n t s c a r r i e d o u t w i t h a f f i n i t y g e l p a r t i c l e s i n suspension.
In the
l a t t e r e x p e r i m e n t s , 3-ml volumes of enzyme s o l u t i o n s a t s p e c i f i e d enzyme c o n c e n t r a t i o n s and i o n i c c o m p o s i t i o n s
were mixed w i t h 0.5 m l o f g e l t h a t had p r e v i o u s l y been packed i n a column and washed i n t h e c o n t a c t i n g b u f f e r . The enzyme-gel s u s p e n s i o n s were mixed f o r t w o h o u r s , t h e g e l w a s removed by c e n t r i f u g a t i o n , and t h e s u p e r n a t a n t w a s a s s a y e d f o r enzyme a c t i v i t y .
The mixing w a s
then continued u n t i l s e q u e n t i a l assays o f t h e supern a t a n t i n d i c a t e d a c o n s t a n t c o n c e n t r a t i o n o f enzyme. The c o n c e n t r a t i o n o f enzyme bound t o t h e g e l p a r t i c l e s
w a s determined b y m a t e r i a l balance. E s t i m a t i o n of G e l P o r e Volume Bovine serum albumin (BSA, m o l e c u l a r w e i g h t 6 8 , 0 0 0 ) w a s added t o 0.5 m l o f g e l t h a t had been packed
i n a column and washed w i t h b u f f e r .
was made up t o o n e m l w i t h b u f f e r .
The t o t a l volume After s t i r r i n g for
1 5 minutes, t h e m i x t u r e w a s c e n t r i f u g e d a t low s p e e d t o s e d i m e n t t h e g e l t o i t s o r i g i n a l packed volume, and t h e p r o t e i n c o n c e n t r a t i o n o f t h e s u p e r n a t a n t was d e t e r m i n e d
4 86
SWART2 ET AL.
by i t s absorbance a t 280 nm.
S i n c e BSA p e n e t r a t e s t h e
p o r e s of t h e g e l , t h e c o n c e n t r a t i o n (C) of BSA i s given by:
M
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=
vp + VI + vs
where M i s t h e mass of p r o t e i n added, and t h e denominat o r i s t h e t o t a l volume a v a i l a b l e t o p r o t e i n [ t h e sum o f t h e pore volume i n t h e g e l p a r t i c l e s (V,),
the inter-
s t i t i a l volume between t h e p a r t i c l e s i n t h e packed g e l l a y e r (V,)
, and
The value o f (Vp
t h e s u p e r n a t a n t volume (Vs = 0.5 m l ) 1.
+ V,)
was determined t o b e 0.32 m l i n
0.5 m l of packed g e l .
The t o t a l volume o f l i q u i d , excluding t h e pore vo 1i n a suspension i d e n t i c a l t o t h e above was
ume (V,),
measured by u s i n g b a k e r ' s y e a s t i n s t e a d of BSA.
The
diameter of y e a s t i s approximately 5 um, and t h e organ-
isms cannot p e n e t r a t e t h e g e l pores, which have a s i z e l i m i t of approximately 0.02 s o n a l communication). pension gel.
(lo7
u m (P.L.
Biochemicals, per-
An a l i q u o t ( 0 . 3 m l ) of y e a s t sus-
cells/ml) w a s added t o 0.5 m l o f packed
A f t e r t h e t o t a l volume w a s a d j u s t e d t o one m l ,
t h e g e l was stirred f o r 15 minutes and c e n t r i f u g e d a t low speed.
The y e a s t c o n c e n t r a t i o n i n t h e s u p e r n a t a n t
w a s then determined with a hemocytometer, and Vs w a s c a l c u l a t e d t o be 0.63 ml.
+ VI
Therefore, VI was 0.13
m l (26% o f t h e packed g e l volume) and Vp w a s 0.19 m l
487
ACETATE KINASE ( 3 8 % o f t h e packed g e l volume).
T h i s v a l u e o f Vp was
used t o c a l c u l a t e t h e c o n c e n t r a t i o n [ E L I o f enzymel i g a n d complex i n t h e p o r e s o f t h e g e l . RESULTS
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P u r l f i c a t i on o f Acetate K i n a s e The p a r t i a l l y p u r i f i e d commercial acetate k i n a s e p r e p a r a t i o n was a d s o r b e d by g e l t y p e s 2 and 3 , i n which AMP i s bound t h r o u g h t h e a d e n i n e r i n g , b u t n o t by g e l
i n which AMP i s bound t h r o u g h t h e r i b o s e group.
type 4 ,
These o b s e r v a t i o n s s u g g e s t t h a t t h e s p e c i f i c i t y of t h e enzyme t o w a r d s a d e n i n e n u c l e o t i d e s is n o t m a n i f e s t e d t h r o u g h t h e a d e n i n e moiety.
The a b i l i t y of acetate k i n -
a s e t o u s e guanosine 5 ' - t r i p h o s p h a t e 5'-triphosphate action'
'
(GTP) and i n o s i n e
( I T P ) as s u b s t r a t e s i n t h e r e v e r s e re-
is i n accordance w i t h t h i s i n t e r p r e t a t i o n .
G e l type 3 exhibited a s t r o n g e r a f f i n i t y €or acetate
k i n a s e t h a n d i d g e l t y p e 2 and w a s u s e d i n a l l subseq u e n t experiments. F i g . 1 shows t h e s p e c i f i c a d s o r p t i o n and e l u t i o n from t y p e 3 g e l o f t h e p a r t i a l l y p u r i f i e d acetate k i n a s e from Sigma.
When BSA w a s added t o t h e c o n t a c t -
i n g s o l u t i o n , v i r t u a l l y a l l o f i t passed through t h e column, w h e r e a s acetate k i n a s e w a s r e t a i n e d and w a s e l u t e d by 1 0 mM ADP b u t n o t by an N a C l s o l u t i o n o f comparable i o n i c s t r e n g t h .
The p r e s e n c e o f Mg2+ and
a low i o n i c s t r e n g t h w a s b e n e f i c i a l f o r b i n d i n g t h e
SWART2 ET AL.
488
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1 2 0 1 ,
0
I
10
,
I
,
,
I
20 30 EFFLUENT VOLUME ( m l )
,6
40
FIGURE 1
A f f i n i t y chromatography of p a r t i a l l y p u r i f i e d acet a t e k i n a s e (Sigma) on a n agarose-bound C 8 - (6-aminohexy1)-AMP g e l . The column w a s 6 x 0 . 9 c m , and t h e A. E i g h t e e n m l o f soluflow r a t e was 0 . 4 ml/minute. t i o n c o n t a i n i n g a c e t a t e k i n a s e ( 2 2 U / m l ) , 1 0 mM MgC12, 1 mM DTT, and 4 mg/ml BSA i n 0 . 1 M phosphate b u f f e r a t pH 7.4 were a p p l i e d t o t h e column a t room t e m p e r a t u r e . B. Wash s o l u t i o n : 1 mM DTT and 1 0 mM MgC12 a t pH 7.4. C. E l u t i n g s o l u t i o n : same a s (B) b u t w i t h 1 0 mM ADP. S p e c i f i c a c t i v i t y of t h e enzyme p r i o r t o a d d i t i o n of BSA w a s 930 U/mg; a f t e r a d d i t i o n o f BSA i t w a s 5.5 U/mg. Of t h e 4 0 0 U o f enzyme added t o t h e column, 222 U were bound and recovered on e l u t i o n . S p e c i f i c a c t i v i t y of t h e recovered enzyme was 6 0 0 0 U/mg p r o t e i n .
enzyme t o t h e g e l .
The s p e c i f i c a c t i v i t y of t h e p u r i -
f i e d enzyme w a s 6 , 0 0 0 U p e r mg p r o t e i n , which r e p r e s e n t s a s i x - f o l d p u r i f i c a t i o n over t h e DCT-activated enzyme a p p l i e d t o t h e column ( s p e c i f i c a c t i v i t y 930 U p e r mg p r o t e i n ) .
The p u r i f i c a t i o n o f acetate k i n a s e t o
such a h i g h d e g r e e h a s n o t been r e p o r t e d p r e v i o u s l y . The h i g h e s t s p e c i f i c a c t i v i t i e s i n t h e l i t e r a t u r e r a n g e from 1 6 0 t o 300 U p e r mg p r o t e i n .
I
Polyacrylamide
g e l e l e c t r o p h o r e s i s of t h e p u r i f i e d enzyme i s o l a t e d from t h e Sigma p r e p a r a t i o n r e v e a l e d o n l y a s i n g l e band.
489
ACETATE KINASE
The absence o f DTT d u r i n g e l e c t r o p h o r e s i s , however, r e s u l t e d i n one l a r g e d i f f u s e band.
T h i s phenomenon
p r o b a b l y a r i s e s from t h e p r e s e n c e of b o t h reduced and o x i d i z e d forms o f t h e enzyme.
Assays o f g e l s e c t i o n s
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soaked i n DTT s o l u t i o n showed enzyme a c t i v i t y througho u t t h e s t a i n e d zone. The a d s o r p t i o n - e l u t i o n p r o f i l e o f t h e c r u d e , c e l l f r e e e x t r a c t prepared from E . c o l i u s i n g t y p e 3 g e l i s shown i n Fig.
2.
Adenylate k i n a s e , which i n t e r f e r e s i n
t h e acetate k i n a s e a s s a y system, showed no a f f i n i t y f o r the gel.
The s p e c i f i c a c t i v i t y of t h e acetate k i n a s e
EFFLUENT VOLUME Im l 1
FIGURE 2 A f f i n i t y chromatography o f a c r u d e e x t r a c t o f E. The column of F i g . 1 w a s used, b u t t h e f l o w r a t e was 0 . 1 7 ml/minute. A. Nineteen m l o f c r u d e e x t r a c t i n 0 . 1 M KH2P04, 1 mM DTT, and 1 0 mM MgC12 were a p p l i e d . E n t e r i n g enzyme c o n c e n t r a t i o n s were 2 . 2 U / m l f o r adenyla t e k i n a s e and 3 2 . 3 U / m l f o r acetate k i n a s e ( s p e c i f i c a c t i v i t y 1 4 U/mg p r o t e i n ) . B. Wash s o l u t i o n : 1 mM DTT and 1 mM MgC12. C. E l u t i o n by 1 mM DTT, 1 mM MgC12, and 1 0 mM ADP. O f t h e 6 1 4 U of a c e t a t e k i n a s e a p p l i e d , 3 6 8 were r e t a i n e d by t h e column, and 3 3 5 U ( 9 1 % o f t h o s e bound) were r e c o v e r e d a t a s p e c i f i c a c t i v i t y of 5 3 0 U/mg.
coli.
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490
e l u t e d w i t h 1 0 mM ADP w a s 530 U p e r mg p r o t e i n , which r e p r e s e n t s a 38-fold p u r i f i c a t i o n from t h e crude extract.
Although d i s c g e l e l e c t r o p h o r e s i s r e v e a l e d
s e v e r a l p r o t e i n bands , t h i s p r e p a r a t i o n c o n t a i n e d no
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p r o t e a s e , phosphatase, o r a d e n y l a t e k i n a s e a c t i v i t i e s and produced t h e same e q u i l i b r i u m n u c l e o t i d e concentrat i o n s a s d i d t h e acetate k i n a s e o b t a i n e d from Sigma, thereby i n d i c a t i n g t h e absence o f s i g n i f i c a n t A T P a s e activity. The enzyme a c t i v a t i o n observed upon t h e a d d i t i o n of DTT i s c o n s i s t e n t w i t h t h e e x i s t e n c e o f a c t i v e and
i n a c t i v e forms o f t h e enzyme.
To d e t e r m i n e whether t h e
i n a c t i v e form can b i n d t o t h e column, a p a r t i a l l y reacti v a t e d enzyme s o l u t i o n was a p p l i e d u n t i l t h e column approached s a t u r a t i o n .
The enzyme s o l u t i o n n o t r e t a i n -
e d by t h e g e l w a s c o l l e c t e d , and t h e column w a s t h e n e l u t e d w i t h 1 0 mM ADP t o form a second s o l u t i o n . s o l u t i o n s were e q u i l i b r a t e d w i t h 1 mM DTT.
Both
After 24
h o u r s , t h e a c t i v i t y o f t h e enzyme n o t r e t a i n e d had doubled, while t h a t o f t h e enzyme bound and t h e n e l u t e d had n o t i n c r e a s e d .
These r e s u l t s s u g g e s t t h a t o n l y
c a t a l y t i c a l l y a c t i v e enzyme i s r e t a i n e d by t h e i m b i l i z e d AMP.
E q u i l i b r i u m Binding S t u d i e s The a d s o r p t i o n p r o c e s s may be expressed by: E + L C E L
(2)
where E is t h e f r e e enzyme i n s o l u t i o n , L i s t h e l i g a n d
ACETATE KINASE
491
(AMP) a t t a c h e d t o t h e a f f i n i t y g e l , and EL i s t h e imb i l i z e d enzyme-ligand complex.
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tion constant
(%)
An e q u i l i b r i u m a s s o c i a -
can be d e f i n e d a s :
Adsorption i s o t h e r m s measured w i t h t h e Sigma ace-
t a t e k i n a s e p r e p a r a t i o n and t h e a f f i n i t y g e l i n column and s u s p e n s i o n experiments a r e shown i n Fig.
3.
Ident-
i c a l l i n e a r i s o t h e r m s d e s c r i b e d t h e amount of enzyme adsorbed d u r i n g b o t h column and s u s p e n s i o n e x p e r i m e n t s , and t h e s l o p e of t h e l i n e gave t h e v a l u e o f t h e p r o d u c t K [ L ] , which i s a measure o f t h e enzyme b i n d i n g capaL
c i t y of t h e gel.
Although b o t h enzyme p r e p a r a t i o n s
were a p p l i e d t o t h e column w i t h s o l u t i o n s o f t h e same composition, t h e s l o p e f o r t h e Sigma p r e p a r a t i o n w a s 8.5 and t h a t f o r t h e E. c o l i e x t r a c t 5.6.
This d i f f e r -
e n c e may b e due t o d i f f e r e n t l e v e l s of i m p u r i t i e s i n t h e two p r e p a r a t i o n s .
Column s i z e and geometry, f l o w
r a t e , r e s i d e n c e t i m e , t o t a l armunt of enzyme c o n t a c t e d , and t h e enzyme c o n c e n t r a t i o n i n the e n t e r i n g s o l u t i o n
w e r e varied.
The p r o d u c t K L [ L ] c o r r e l a t e d o n l y w i t h
t h e c o n c e n t r a t i o n o f enzyme s o l u t i o n a p p l i e d t o t h e column. The l i n e a r i t y of t h e p l o t s i n d i c a t e s t h a t K L I L l
w a s c o n s t a n t , and s u g g e s t s t h a t t h e e f f e c t i v e l i g a n d c o n c e n t r a t i o n was n o t s i g n i f i c a n t l y reduced by t h e
SWART2 ET AL.
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492
FIGURE 3 Adsorption isotherms obtained from column purifications and equilibrium experiments with gel suspensions in 0.1 M KH2P04, 10 mM Mg++, and 1 mM DTT. Acetate kinase (Sigma): 0 , gel suspension experiment, 0 , column experiment; crude E. coli extract: A, gel suspension experiment, A , column experiment. In the gel suspension experiments, [El is the equilibrium concentration of enzyme in the supernatant, and [EL] is the quantity of enzyme adsorbed divided by the calculated gel pore volume; in the column purification experiments, [El is the enzyme concentration in the inlet stream, and [EL] is calculated from the amount of enzyme retained on the column.
ACETATE KINASE
493
enzyme a d s o r p t i o n .
T h i s f i n d i n g i s s u p p o r t e d by an est-
imated maximum g e l c a p a c i t y of 4.2
x lo6 U p e r m i of
pore volume ( c a l c u l a t e d from 5.8 p m o l i m m b i l i z e d AMP p e r m l of packed g e l ; m o l e c u l a r weight 4 1 , 0 0 0 f o r ace-
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t a t e k i n a s e from E . c o l i 1 2 ; 6,000 U/mg f o r t h e p u r i f i e d p r o t e i n ; one enzyme bound t o each A M P ) , which i s approximately
lo5
t i m e s as g r e a t as t h e h i g h e s t c o n c e n t r a t i o n
of enzyme used.
Thus, even i f o n l y a v e r y s m a l l f r a c -
t i o n of t h e AMP w e r e a c c e s s i b l e , i t would s t i l l be i n g r e a t excess compared t o t h e q u a n t i t i e s of enzyme adsorbed. E f f e c t s o f Magnesium I o n C o n c e n t r a t i o n and Ionic Strength E a r l y experiments s u g g e s t e d t h a t magnesium i o n conc e n t r a t i o n and i o n i c s t r e n g t h e x e r t major i n f l u e n c e s on g e l c a p a c i t y .
The e f f e c t s o f b o t h w e r e t h e r e f o r e
e x p l o r e d i n an a t t e m p t t o m a x i m i z e K L [ L ] .
Fig. 4 p r e -
s e n t s v a l u e s o f K L [ L ] as a f u n c t i o n o f i o n i c s t r e n g t h a t two c o n c e n t r a t i o n s of M g C 1 2 .
Data a t t h e two high-
e s t i o n i c s t r e n g t h s were t a k e n from g e l s u s p e n s i o n experiments.
The o t h e r d a t a were o b t a i n e d from t h e
column wash s t e p o f p u r i f i c a t i o n s o f t h e Sigma enzyme preparation.
++
Wash s o l u t i o n s c o n t a i n i n g t h e same Mg
c o n c e n t r a t i o n b u t d i f f e r i n g i n N a C l c o n c e n t r a t i o n s were a p p l i e d t o a column c o n t a i n i n g a d s o r b e d enzyme u n t i l t h e enzyme c o n c e n t r a t i o n o f t h e e f f l u e n t r e a c h e d a c o n s t a n t v a l u e , which w a s used t o estimate K L [ L ] .
SWART2 ET AL.
494
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0
60
120 180 240 I O N I C STRENGTH I m M )
300
FIGURE 4 KL[L] as a f u n c t i o n o f i o n i c s t r e n g t h . Except f o r t h e d a t a of t h e t w o highest i o n i c s t r e n g t h s , values of K L [ L ] were o b t a i n e d d u r i n g column p u r i f i c a t i o n s by d e t e r m i n i n g t h e enzyme c o n c e n t r a t i o n i n t h e wash s o l u The t i o n as i t s c o n c e n t r a t i o n of N a C l was i n c r e a s e d . s o l u t i o n s a s a p p l i e d c o n t a i n e d o n l y NaC1, 1 mM DTT, a n d t h e c o n c e n t r a t i o n o f MgC12 i n d i c a t e d (0, 6 0 mM; 0 , 1 0 mM) KL [ L ] was computed w i t h [ E l a s t h e a c e t a t e k i n a s e c o n c e n t r a t i o n i n t h e e f f l u e n t a t s t e a d y s t a t e and [EL] a s t h e c a l c u l a t e d c o n c e n t r a t i o n o f i m m o b i l i z e d enzyme.
.
S i n c e enzyme c o n c e n t r a t i o n s i n t h e e f f l u e n t w e r e n o t s e n s i t i v e t o l i q u i d f l o w rates, w e c o n c l u d e d t h a t t h e enzyme c o n c e n t r a t i o n s were c o n t r o l l e d b y t h e enzymeligand equilibrium.
A s l i g h t increase i n t h e ionic
s t r e n g t h of t h e s o l u t i o n c a u s e d a l a r g e decrease i n t h e measured v a l u e of K L I L I a t b o t h c o n c e n t r a t i o n s of MgC12. The s l o p e s of t h e l i n e s i n Fig. a v e r a g e v a l u e of -10.9.
4 are s i m i l a r , w i t h a n
The v a r i a t i o n i n K L I L l w i t h
i o n i c s t r e n g t h may be e x p r e s s e d by: A ( l 0 g K L I L I ) = -10.9Ap
(4)
where p i s t h e i o n i c s t r e n g t h . KL[L] w a s n e x t measured w i t h t h e Sigma a c e t a t e k i n -
ase p r e p a r a t i o n a t i n c r e a s i n g c o n c e n t r a t i o n s of MgC12.
49 5
ACETATE KINASE
Because o f t h e a p p r e c i a b l e c o n t r i b u t i o n by MgCl, t o t h e i o n i c s t r e n g t h , no a t t e m p t w a s made t o k e e p i o n i c s t r e n g t h constant.
To a v o i d f o r m a t i o n o f magnesium
p h o s p h a t e p r e c i p i t a t e s , measurements were made w i t h
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enzyme-gel buffer.
suspensions with T r i s r a t h e r than phosphate
A s shown i n F i g .
5 , KLILI i n c r e a s e d r a p i d l y
FIGURE 5 Dependence o f KL [ L l on MgC12. V a l u e s were o b t a i n e d from e q u i l i brium experiments w i t h Sigma a c e t a t e k i n a s e and a f f i n i t y g e l s u s p e n s i o n s i n 1 mM DTT (0, i n 0 . 1 M T r i s b u f f e r ; 0 , i n 0.01 M T r i s ) . The maximum i n K L [ L ] o c c u r r e d a t [MgC12 I o f 60 mM w i t h 0 . 0 1 M T r i s and of 4 0 mM w i t h 4 0 mM T r i s .
496
SWARTZ ET AL.
w i t h i n c r e a s i n g c o n c e n t r a t i o n s of M g C 1 2 , r e a c h e d a peak, and t h e n d e c l i n e d . Equation 4 w a s a p p l i e d t o t h e r e s u l t s i n Fig. 5 t o s e p a r a t e t h e e f f e c t s r e s u l t i n g from c h a n g e s i n i o n i c
++ I .
The con-
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s t r e n g t h from t h o s e due t o changes i n [Mg
d i t i o n s c o r r e s p o n d i n g t o t h e maximum v a l u e i n Fig. 5 (KL[L] = 58,
[MgC12] = 6 0 mM,
p = 263 mM) were s e l e c t e d
as a b a s i s €or comparison, and Ap f o r e a c h d a t a p o i n t was t a k e n t o b e t h e d i f f e r e n c e between t h e a c t u a l i o n i c s t r e n g t h and 0.268 M.
The v a l u e o f A(1og K L I L I ) w a s
t h e n c a l c u l a t e d from E q u a t i o n 4 and added t o t h e logarithm of t h e KL[L] v a l u e a c t u a l l y measured.
Fig.
6 is
++ I
a p l o t of t h e c a l c u l a t e d v a l u e of K L I L I v e r s u s [Mg
t h a t would have been o b t a i n e d i f i t were p o s s i b l e t o m a i n t a i n t h e i o n i c s t r e n g t h c o n s t a n t a t 0.268 out.
M through-
The two s e p a r a t e c u r v e s o b t a i n e d i n c r e a s e d mono-
t o n i c a l l y w i t h [MgC12] and c o r r e s p o n d e d t o t h e two c o n c e n t r a t i o n s o f T r i s b u f f e r used. To show t h a t t h e r e s u l t s o f t h e enzyme-gel suspens i o n c o n t a c t s i n Fig.
5 a l s o a p p l i e d t o column p u r i f i c a -
t i o n s , a n experiment w a s performed w i t h t h e Sigma enzyme under o p t i m a l c o n d i t i o n s f o r a d s o r p t i o n . f i v e m l of enzyme s o l u t i o n ( 5 . 3 9 U / m l )
Sixty-
i n 60 mM MgC12,
26 mM (NH4)2S04, 3 mM DTT, and 0 . 0 1 M T r i s a t pH 7.4 ( p = 0.263 M ) were a p p l i e d t o a column c o n t a i n i n g 2 m l
of g e l (Vp = 0.76 m l ) .
Of t h e 350 U a p p l i e d , 240 w e r e
ACETATE KINASE
49 7
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'o*ooo*
0
20
40 [MpCI,]
60 80 (mM)
100
FIGURE 6 The e f f e c t o f [MgCl21 on K L [ L ] when c o r r e c t e d f o r t h e e f f e c t of i o n i c s t r e n g t h . Equation 4 was used t o a d j u s t measured values of KL[LI t o t h o s e t h a t would b e e x p e c t e d i f p had been c o n s t a n t a t 0 . 2 6 8 M. 0, i n 0.1 M T r i s buffer; 0 , i n 0.01 M T r i s .
r e t a i n e d by t h e g e l .
T h i s compares w e l l w i t h t h e capa-
c i t y e x p e c t e d on t h e b a s i s o f t h e g e l s u s p e n s i o n e x p e r i ments ( K L [ L ] [ E ] V p = 238 U).
When t h e column w a s e l u t e d
w i t h 10 m l of a s o l u t i o n c o n t a i n i n g 1 0 mM ADP, 6 0 mM MgC12, and 1 mM DTT, a d j u s t e d t o pH 7.4, 205
u
(85%)
were r e c o v e r e d a t a n a v e r a g e s p e c i f i c a c t i v i t y of 2155 U p e r mg p r o t e i n .
These r e s u l t s r e p r e s e n t a s i g n i f i -
c a n t improvement i n performance i n comparison t o t h o s e o f F i g . 1, wherein 222 U were r e t a i n e d by 4 r n l o f
SWART2 ET AL.
49 8
g e l a t a n e n t e r i n g enzyme c o n c e n t r a t i o n of 2 2 U/ml.
If
t h e experiment of F i g . 1 had been conducted w i t h a g e l volume of 2 m l and an e n t e r i n g enzyme c o n c e n t r a t i o n of 5.39 U / m l ,
o n l y 27 U would have been e x t r a c t e d from t h e
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entering solution.
Although t h e p r o d u c t o f t h e f i n a l
experiment w a s n o t a s p u r e as t h a t o b t a i n e d i n t h e experiment shown i n F i g . 1 (2155 vs. 6 0 0 0 U/mg p r o t e i n ) , t h e column c a p a c i t y h a s been i n c r e a s e d n i n e - f o l d .
This
i n c r e a s e may b e a t t r i b u t e d t o t h e i n c r e a s e i n [MgCl21 from 1 0 t o 60 mM and t h e d e c r e a s e i n i o n i c s t r e n g t h from 331 t o 263 mM. D I S CUSSION
W e have shown t h a t AMP, bound t h r o u g h i t s a d e n i n e moiety t o a g a r o s e , a c t s as an e f f e c t i v e l i g a n d f o r t h e b i o s p e c i f i c a d s o r p t i o n o f acetate k i n a s e .
Partially
p u r i f i e d acetate k i n a s e o b t a i n e d from Sigma was p u r i f i e d t o a p p a r e n t homogeneity by t h i s method.
By a s i n -
g l e p a s s t h r o u g h t h e a f f i n i t y column, a c e l l - f r e e ex-
t r a c t from E. c o l i was p u r i f i e d t o a d e g r e e s u i t a b l e f o r u s e i n a l a r g e - s c a l e enzyme r e a c t o r ( a b s e n c e of p r o t e a s e , p h o s p h a t a s e and A T P a s e a c t i v i t i e s )
.
The a d s o r p t i o n i s o t h e r m s were l i n e a r w i t h b o t h t h e Sigma enzyme p r e p a r a t i o n and t h e c r u d e E . c o l i e x t r a c t , and y i e l d e d KLILI v a l u e s of 9.4 and 6.3, i n 0 . 1 M phosphate (pH 7 . 4 )
respectively,
and 1 0 mM Mg++.
The d i f f e r -
ence i n s l o p e s may be a t t r i b u t e d t o t h e h i g h e r level of Contaminant p r o t e i n s i n t h e crude e x t r a c t , which would
499
ACETATE KINASE
i n c r e a s e t h e i o n i c s t r e n g t h o f t h e s o l u t i o n , r e d u c e by
++
c h e l a t i o n t h e e f f e c t i v e Mg
c o n c e n t r a t i o n , and p o s s i -
b l y compete f o r t h e l i g a n d . Both t h e i o n i c s t r e n g t h o f t h e s o l u t i o n and t h e
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m a gne s ium i o n concen tr a t i on s t r o n q l y in f 1uen ced acetate k i n a s e adsorption.
The v e r y pronounced i n c r e a s e i n
KL ILI w i t h d e c r e a s i n g i o n i c s t r e n g t h w a s unexpected,
b u t t h e r e s u l t s a r e c o n s i s t e n t w i t h r o u t i n e l y observed column performance, t h a t i s , when t h e columns were washed w i t h s o l u t i o n s of a lower i o n i c s t r e n g t h t h a n t h a t o f t h e enzyme s o l u t i o n s a p p l i e d , v e r y l i t t l e loss o f enzyme from t h e column was observed.
I f t h e lower
i o n i c s t r e n g t h had n o t caused a d r a m a t i c i n c r e a s e i n t h e e q u i l i b r i u m c o n s t a n t f o r a d s o r p t i o n , and K L [ L ] had remained t h e same, t h e n t h e e x i t i n g enzyme c o n c e n t r a t i o n d u r i n g t h e wash s t e p f o l l o w i n g s a t u r a t i o n o f t h e column would have been e q u a l t o t h e c o n c e n t r a t i o n o f t h e enzyme s o l u t i o n o r i g i n a l l y a p p l i e d .
The i n s i g n i f i -
c a n t l o s s of enzyme r o u t i n e l y observed d u r i n g t h e wash s t e p o f column p u r i f i c a t i o n s under t h e s e c o n d i t i o n s i s p o s s i b l e o n l y w i t h v e r y h i g h v a l u e s of K L I L I .
The
dependence o f KLILI on i o n i c s t r e n g t h is a l s o i n q u a l i t a t i v e agreement w i t h t h e o b s e r v a t i o n s of Robinson
a1.,13
et
who found t h a t t h e b i n d i n g of 6 - g a l a c t o s i d a s e
t o an a f f i n i t y matrix decreased s u b s t a n t i a l l y w i t h i n c r e a s i n g i o n i c s t r e n g t h of t h e b u f f e r .
SWART2 ET AL.
500
The s t r o n g dependence of t h e g e l c a p a c i t y on
++ [Mg ]
c a n n o t b e e x p l a i n e d s i m p l y by t h e i n c r e a s e d
++ 3 .
MgAMP c o n c e n t r a t i o n s w i t h i n c r e a s i n g [ M g
The i n -
crease i n [MgAMP] i s a t most a f i r s t o r d e r f u n c t i o n o f
++ 1, ++ [Mg I
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[Mg
whereas KL [L] i n c r e a s e s l o g a r i t h m i c a l l y w i t h i n Fig. 6.
t o i n c r e a s e KL.
Mg++ may a c t d i r e c t l y on t h e enzyme I t i s a l s o p o s s i b l e t h a t h i g h local
c o n c e n t r a t i o n s o f u n c h e l a t e d AMP (which e x i s t s p r e d o w i n a n t l y a s AMP2' charged g e l .
a t pH 7 . 4 ) would produce a n e g a t i v e l y
I t h a s been shown t h a t i n s o l u t i o n s o f
low i o n i c s t r e n g t h , t h e pH i n t h e microenvironment n e a r t h e g e l can b e s u b s t a n t i a l l y lower t h a n t h a t o f the bulk solution.14
++ 1,
The e f f e c t o f i n c r e a s i n g [Mg
and t h u s [MgAMP], i s t o d e c r e a s e t h e l o c a l n e g a t i v e charge, t h e r e b y i n c r e a s i n g t h e pH i n t h e microenvironment o f t h e l i g a n d and p e r h a p s c h a n g i n g enzyme a f f i n i t y f o r t h e ligand.15
Our r e s u l t s have i m p o r t a n t i m p l i c a t i o n s f o r t h e o p e r a t i o n and s c a l e - u p of enzyme p u r i f i c a t i o n p r o c e s s e s u t i l i z i n g b i o s p e c i f i c adsorption.
A l l three steps
(enzyme a d s o r p t i o n , removal of c o n t a m i n a n t s , and enzyme e l u t i o n ) are a f f e c t e d by t h e i n t e r a c t i o n between enzyme and l i g a n d .
A l a r g e r g e l c a p a c i t y may be o b t a i n e d by
i n c r e a s i n g KL [L] and/or
[El
.
Concentration o f t h e
c r u d e enzyme s o l u t i o n may t h u s be advantageous i f i o n i c s t r e n g t h i s n o t s i g n i f i c a n t l y a f f e c t e d and i f v i s c o s i t y
ACETATE KINASE
501
i s n o t i n c r e a s e d s u c h t h a t f l o w t h r o u g h t h e column becomes d i f f i c u l t .
The v a l u e o f K L [ L ] may b e i n c r e a s e d
++
s e v e r a l - f o l d by a d j u s t i n g i o n i c s t r e n g t h and Mg However, as i n d i c a t e d by Robinson
centration.
con-
% &.13
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and by o u r d a t a , p r o d u c t p u r i t y may d e c r e a s e a s i o n i c s t r e n g t h o f t h e c r u d e s o l u t i o n i s d e c r e a s e d and more non-specific
a d s o r p t i o n occurs. ACKNOWLEDGEMENT
T h i s s t u d y w a s s u p p o r t e d by Grant No. GI34284 from t h e N a t i o n a l S c i e n c e Foundation. REFERENCES AND NOTES 'Present address:
T a t e and L y l e Ltd.,
Reading,
England. 2 Pre sent address:
Amicon C o r p . , L e x i n g t o n , MA.
3T0 whom correspondence s h o u l d be a d d r e s s e d .
4R.S.
Langer, B.K.
and C.K.
Hamilton, C.R.
Colton, Am.
Inst.
Gardner, M.C.
Chem. Eng.,
22,
Archer
1079
(1976). 51.A.
Rose, M.
Grunberg-Manago,
Ochoa, J. B i o l . Chem., 6 ~ . S . Anthony and L.B. 6129-6135 'R.S.
211,
S.R.
737-756
Korey and S . (1954).
S p e c t o r , J. B i o l .
Chem.,
246,
(1971).
Langer, "Enzymatic R e g e n e r a t i o n o f ATP,"
Sc.D.
T h e s i s , M a s s a c h u s e t t s I n s t i t u t e o f Technology, Cambridge, MA, 1974. 80.H.
Lowry, N.J.
Rosebrough, A.L.
R a n d a l l , J. B i o l . Chem.,
F a r r and R.
193, 265-275
(1951).
J.
SWART2 ET AL.
502
90. G a b r i e l , i n "Methods i n Enzymology," Vol. 22, W.B.
Jakoby, ed., Academic, N e w York, 1971, pp.
568-
578. 1°C.H.
F i s k e and Y.
Subbarow, J . B i O l .
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(1925). llC.A.
Janson and W.W.
Cleland, J. B i o l .
Chem.,
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2567-2571 (1974). 12R.S. Anthony a n d L.B.
S p e c t o r , J. B i o l .
Chem.,
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6739 (1970). 13P.J.
Robinson, P . D u n n i l l a n d M.D.
Biophys. A c t a ,
285,
14L. G o l d s t e i n , Y .
try, 15D.L.
149, -
-3,
L i l l y , Biochim.
28-35 (1972).
Levin and E. K a t c h a l s i , Biochemis-
1913-1919 (1964).
P u r i c h and H . J .
307 (1972).
F r o m , Arch. Biochem. Biophys.,
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification of Acetate Kinase by Affinity Chromatography a
James R. Swartz , Gary W. Pace A. Solomon Archer
a c
a b
, Barry
a
, Clark K. Colton & Michael C.
a
a
Department of Nutrition and Food Science (JRS, GWP, MCA) and Department of Chemical Engineering (BAS, CKC) , Massachusetts Institute of Technology , Cambridge, Massachusetts, 02139 b
Tate and Lyle Ltd. , Reading, England
c
Amicon Corp. , Lexington, MA. Published online: 05 Dec 2006.
To cite this article: James R. Swartz , Gary W. Pace , Barry A. Solomon , Clark K. Colton & Michael C. Archer (1978) Purification of Acetate Kinase by Affinity Chromatography, Preparative Biochemistry, 8:6, 479-502, DOI: 10.1080/00327487808061665 To link to this article: http://dx.doi.org/10.1080/00327487808061665
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PREPARATIVE BIOCHEMISTRY, 8(6), 479-502 (1978)
PURIFICATION O F ACETATE KINASE BY AFFINITY CHROMATOGRAPHY
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James R. S w a r t z , Gary W. C l a r k K.
Pace,'
B a r r y A. S o l o m o n I 2
C o l t o n a n d M i c h a e l C. A r c h e r 3
Department o f N u t r i t i o n and Food S c i e n c e (JRS, GWP, MCA) and Department o f Chemical E n g i n e e r i n g (BAS, C K C ) , Mas sac hu s et t s I n s ti t u t e o f Techno 1og y , Cambridge, M a s s a c h u s e t t s
02 1 3 9
ABSTRACT A one-step procedure u s i n g a f f i n i t y chromatography
h a s b e e n shown t o p u r i f y t o a p p a r e n t homogeneity ace-
t a t e k i n a s e from a c o m m e r c i a l l y a v a i l a b l e p r e p a r a t i o n and t o p a r t i a l l y p u r i f y t h e enzyme from a c r u d e , c e l l f r e e extract.
S i n c e t h e g e l ' s c a p a c i t y f o r enzyme ad-
s o r p t i o n i s c o n t r o l l e d by t h e thermodynamics o f l i g a n d enzyme i n t e r a c t i o n , m a x i m i z a t i o n o f t h e a d s o r p t i o n isotherm w a s attempted.
Enzyme a d s o r p t i o n d e c r e a s e d
l o g a r i t h m i c a l l y w i t h i n c r e a s i n g i o n i c s t r e n g t h b u t inc r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f MgC12.
These
c o m p e t i n g e f f e c t s c a u s e d t h e . n e t a d s o r p t i o n of enzyme
479 Copyright 0 1979 hy Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
480
SWART2 ET AL.
t o i n c r e a s e t o a maximum and t h e n t o d e c r e a s e a s t h e MgC12 c o n c e n t r a t i o n was r a i s e d .
The r e s u l t s allow a
s i g n i f i c a n t improvement i n a f f i n i t y column performance and have i m p o r t a n t i m p l i c a t i o n s f o r s c a l e - u p p r o c e d u r e s .
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INTRODUCTION
F i o s y n t h e s i s of many chemicals i n v o l v e s use o f t h e c o f a c t o r a d e n o s i n e t r i p h o s p h a t e (ATP) as a n e n e r g y source.
The economic a p p l i c a t i o n o f enzymes as c a t a -
lysts for industrial-scale synthesis therefore requires t h e development o f a n i n e x p e n s i v e p r o c e s s € o r r e g e n e r a t i o n o f ATP from a d e n o s i n e d i p h o s p h a t e (ADP) o r m n o phosphate (AMP).
The r e l a t i v e merits of v a r i o u s r o u t e s
t o ATP r e g e n e r a t i o n have been e v a l u a t e d 4 ; t h e s e i n v o l v e chemical s y n t h e s i s , whole c e l l s o r o r g a n e l l e s , or c e l l f r e e enzymes. f e r a s e , E.C.
Acetate k i n a s e (ATP :acetate phosphotrans2.7.2.1)
r e g e n e r a t i o n scheme.
i s a key enzyme i n a promising Procedures r e p o r t e d f o r i s o l a t i o n
and p u r i f i c a t i o n o f acetate k i n a s e i n v o l v e a number of s t e p s , i n c l u d i n g p r e c i p i t a t i o n by a c e t o n e and ammonium s u l f a t e , and g e l - and ion-exchange chromatography.
B
I n view of t h e p o t e n t i a l u s e o f acetate k i n a s e f o r ATP r e g e n e r a t i o n , a s i m p l e r and more e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e seems d e s i r a b l e . I n t h e p r e s e n t p a p e r , we d e s c r i b e a s i n g l e - s t e p a f f i n i t y chromatography p r o c e d u r e u s i n g AMP d e r i v a t i v e s of a g a r o s e f o r t h e 38-fold p u r i f i c a t i o n of acetate
4 81
ACETATE KINASE
k i n a s e from a c r u d e , c e l l - f r e e e x t r a c t o f E s c h e r i c h i a
coli.
The r e s u l t i n g p a r t i a l l y p u r i f i e d p r e p a r a t i o n i s
s u i t a b l e f o r d i r e c t u s e i n an ATP-regeneration reactor because it c o n t a i n s n o p r o t e a s e , p h o s p h a t a s e , ATPase o r
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adenylate kinase a c t i v i t i e s .
We a l s o d e s c r i b e t h e p u r i -
f i c a t i o n t o a p p a r e n t homogeneity of a commercially available a c e t a t e kinase preparation. S i n c e t h i s method may f i n d u s e i n i n d u s t r i a l - s c a l e enzyme p r o d u c t i o n , w e have i n v e s t i g a t e d c e r t a i n a s p e c t s of t h e a d s o r p t i o n p r o c e s s t h a t i n f l u e n c e s e p a r a t i o n performance.
The c a p a c i t y o f t h e a f f i n i t y s u p p o r t was
q u a n t i t a t e d i n terms o f t h e p r o d u c t of t h e e f f e c t i v e l i g a n d c o n c e n t r a t i o n and t h e thermodynamic e q u i l i b r i u m c o n s t a n t f o r enzyme-ligand
interaction.
Purification
performance was s i g n i f i c a n t l y enhanced by t h e i d e n t i f ic a t i o n and o p t i m i z a t i o n of p a r a m e t e r s t h a t i n f l u e n c e t h e e q u i l i b r i u m c o n s t a n t f o r enzyme a d s o r p t i o n .
This
i n f o r m a t i o n w i l l be o f v a l u e n o t o n l y f o r i n c r e a s i n g p r o c e s s scale, b u t f o r u n d e r s t a n d i n g t h e thermodynamics and mechanism of a f f i n i t y a d s o r p t i o n methods i n g e n e r a l . MATERIALS AND METHODS
-E. c o l i acetate k i n a s e , y e a s t hexokinase, and g l u cose-6-phosphate
dehydrogenase were o b t a i n e d a s suspen-
s i o n s i n 3 . 2 M ammonium s u l f a t e from Sigma ( S t . L o u i s ) . D i t h i o t h r e i t o l ( DTT) , ADP, AMP, nicotinamide-adenine d i n u c l e o t i d e phosphate (NADP), and a c e t y l phosphate
w e r e a l s o o b t a i n e d from Sigma.
S W T Z ET AL.
482
The a f f i n i t y chromatography g e l s u p p o r t s used i n t h i s s t u d y were o b t a i n e d i n preswollen form from P.L. Biochemicals, I n c .
(Milwaukee), and are d e s i g n a t e d by
t h e manufacturer a s agarose-hexane adenosine 5 I-phos-
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p h a t e (AGAMP) t y p e s 2 , 3 , and 4 .
They c o n s i s t o f AMP
a t t a c h e d t o a n a q a r o s e s u p p o r t by a six-carbon
spacer
through e i t h e r t h e amino group ( t y p e 2 1 , t h e C 8 of t h e adenine moiety ( t y p e 3 ) , o r t h e hydroxyl groups on t h e ribose ring (type 4 ) .
The c o n c e n t r a t i o n o f t h e AMP li-
qand, a s r e p o r t e d by t h e manufacturer, i s 5.8 pmol/ml packed w e t g e l ( t y p e 3 ) . P r e p a r a t i o n of Crude Acetate Kinase
-E. c o l i
(ATCC 9637-1,
w i l d t y p e ) w a s grown anaero-
b i c a l l y a t 37OC on a medium c o n t a i n i n g ( p e r l i t e r ) : 4 q glucose, KH2P04,
1.4
0.5
g ( N H 4 I 2 S O 4 , 0.5
g MgS04, 0.9 g
g N a 2 H P 0 4 , 0.11 mg C a C 1 2 ,
0.22 mg CuSO4,
0.15 mg FeS04, 0 . 2 1 mg Na2Mo04, 0.13 mg CoC12, 0.60 mg MnS04, and 0.81 mg ZnS04.
A crude c e l l - f r e e e x t r a c t
c o n t a i n i n g acetate k i n a s e a c t i v i t y w a s prepared from t h e h a r v e s t e d E. c o l i by r e s u s p e n s i o n of t h e c e l l s i n 0 . 1 M phosphate b u f f e r (pH 7 . 4 1 ,
d i s r u p t i o n i n a Braun
r o t a r y homogenizer, and c e n t r i f u g a t i o n a t 1 5 , 0 0 0 x 9: f o r 1 5 minutes t o remove c e l l d e b r i s .
The s u p e r n a t a n t
c o n t a i n e d approximately 1 5 U acetate k i n a s e p e r m g protein.
This crude e x t r a c t w a s s t o r e d i n l i q u i d n i t r o g e n .
To p r e v e n t l o s s of a c t i v i t y by o x i d a t i o n of s u l f h y d r y l g r o u p s r 7 1 . 0 mM DTT was added t o a l l enzyme s o l u t i o n s .
483
ACETATE KINASE
A d d i t i o n o f DTT t o t h e p a r t i a l l y p u r i f i e d a c e t a t e kin-
ase from Sigma i n c r e a s e d a c t i v i t y from 1 7 0 U p e r mg prot e i n , a s r e p o r t e d by Sigma, o r 2 2 0
u
p e r mg p r o t e i n , a s
measured by o u r a s s a y , t o 930 U p e r mg p r o t e i n .
Since
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t h e commercial enzyme was o b t a i n e d a s a s u s p e n s i o n i n c o n c e n t r a t e d (NH4)2SOqr it w a s c o n v e n i e n t t o a d j u s t a l l enzyme s o l u t i o n s t o a uniform c o n c e n t r a t i o n ( 2 6 mM) of ( N H 4 ) 2so4.
One u n i t of acetate k i n a s e was d e f i n e d as t h e amount o f enzyme t h a t c a t a l y z e s t h e p r o d u c t i o n of one pmol A T P p e r minute when added t o t h e f o l l o w i n g a s s a y solution:
5 mM ADP,
5 mM a c e t y l p h o s p h a t e , 1 0 mM glu-
c o s e , 0.3 mM NADP, 1 0 mM MgC12, 5 U/ml hexokinase, and 2 U/ml
glucose-6-phosphate
b u f f e r a t pH 1 . 4 and 3OOC.
dehydrogenase i n 0.1 M T r i s Formation of NADPH w a s
monitored c o n t i n u o u s l y by s p e c t r o p h o t o m e t r y a t 34 0 nm. Adenylate k i n a s e a c t i v i t y w a s determined by t h e same c o n t i n u o u s a s s a y e x c e p t t h a t a c e t y l phosphate w a s n o t included i n t h e assay solution.
When b o t h enzymes were
p r e s e n t , t h e measured acetate k i n a s e a c t i v i t y w a s c o r r e c t e d by s u b t r a c t i n g from it one h a l f of t h e adenyla t e k i n a s e a c t i v i t y , which w a s less t h a n 1 0 % o f t h e
acetate k i n a s e a c t i v i t y i n t h e c r u d e e x t r a c t and w a s n e g l i g i b l e i n t h e commercial p r e p a r a t i o n . p r o t e i n was monitored e i t h e r by s p e c t r o p h o t o m e t r y a t 280 nm o r by t h e Lowry p r o c e d u r e 8 a f t e r d i a l y s i s t o remove t h e i n t e r f e r i n g DTT.
SWART2 ET AL.
484
D i s c g e l e l e c t r o p h o r e s i s w a s performed according
t o t h e procedure of G a b r i e l g f o r an a n i o n i c enzyme sample (system I , 7.5% acrylamide)
.
I n characterizing
t h e enyzme s o l u t i o n p u r i f i e d from E . c o l i , phosphatase
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a c t i v i t y w a s determined by t h e method of F i s k e and Subbarow.lo
P r o t e a s e a c t i v i t y w a s determined by incub-
a t i n g t h e s o l u t i o n i n c o n t a c t w i t h c a s e i n a t 4OoC f o r one hour.
Any change i n t h e l e v e l o f n o n p r e c i p i t a b l e
p r o t e i n s w a s t h e n a s s a y e d by t h e Lowry procedure.0 Column P u r i f i c a t i o n A l l p u r i f i c a t i o n s were carried o u t a t pH 7.4
and
room temperature, i n a mode p r o p e r l y termed a f f i n i t y o r b i o s p e c i f i c adsorption.
A solution containing the
enzyme t o be p u r i f i e d w a s a p p l i e d t o a column ( 9 mm x 6 c m ) c o n t a i n i n g 2-4
m l o f a f f i n i t y gel.
When t h e
column was s a t u r a t e d (denoted by t h e c o n c e n t r a t i o n of enzyme i n t h e e x i t stream exceeding 90% of t h a t i n t h e i n l e t stream), enzyme a d d i t i o n was terminated.
The
column w a s t h e n washed w i t h 8 m l o f low-ionic s t r e n g t h s o l u t i o n c o n t a i n i n g o n l y 1 mM DTT and a c o n c e n t r a t i o n o f MgC12 e q u a l t o t h a t i n t h e enzyme s o l u t i o n a p p l i e d t o t h e column.
The acetate k i n a s e w a s e l u t e d w i t h a
s o l u t i o n of 1 0 mM ADP and 1 mM DTT, c o n t a i n i n g t h e same c o n c e n t r a t i o n of MgC12.
Sample a l i q u o t s (1-4 m l ) o f
e f f l u e n t were c o l l e c t e d and assayed.
A f t e r each use,
t h e g e l w a s washed w i t h 1 M N a C l t o r e m v e n o n s p e c i f i -
485
ACETATE KINASE
c a l l y bound p r o t e i n and s t o r e d a t 5OC i n 0.02% sodium a z i d e s o l u t i o n u n t i l reuse. Equilibrium Binding S t u d i e s The c a p a c i t y o f t h e g e l f o r enzyme-ligand i n t e r -
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a c t i o n was e v a l u a t e d from t h e r e s u l t s of column p u r i f i c a t i o n experiments and a l s o from e x p e r i m e n t s c a r r i e d o u t w i t h a f f i n i t y g e l p a r t i c l e s i n suspension.
In the
l a t t e r e x p e r i m e n t s , 3-ml volumes of enzyme s o l u t i o n s a t s p e c i f i e d enzyme c o n c e n t r a t i o n s and i o n i c c o m p o s i t i o n s
were mixed w i t h 0.5 m l o f g e l t h a t had p r e v i o u s l y been packed i n a column and washed i n t h e c o n t a c t i n g b u f f e r . The enzyme-gel s u s p e n s i o n s were mixed f o r t w o h o u r s , t h e g e l w a s removed by c e n t r i f u g a t i o n , and t h e s u p e r n a t a n t w a s a s s a y e d f o r enzyme a c t i v i t y .
The mixing w a s
then continued u n t i l s e q u e n t i a l assays o f t h e supern a t a n t i n d i c a t e d a c o n s t a n t c o n c e n t r a t i o n o f enzyme. The c o n c e n t r a t i o n o f enzyme bound t o t h e g e l p a r t i c l e s
w a s determined b y m a t e r i a l balance. E s t i m a t i o n of G e l P o r e Volume Bovine serum albumin (BSA, m o l e c u l a r w e i g h t 6 8 , 0 0 0 ) w a s added t o 0.5 m l o f g e l t h a t had been packed
i n a column and washed w i t h b u f f e r .
was made up t o o n e m l w i t h b u f f e r .
The t o t a l volume After s t i r r i n g for
1 5 minutes, t h e m i x t u r e w a s c e n t r i f u g e d a t low s p e e d t o s e d i m e n t t h e g e l t o i t s o r i g i n a l packed volume, and t h e p r o t e i n c o n c e n t r a t i o n o f t h e s u p e r n a t a n t was d e t e r m i n e d
4 86
SWART2 ET AL.
by i t s absorbance a t 280 nm.
S i n c e BSA p e n e t r a t e s t h e
p o r e s of t h e g e l , t h e c o n c e n t r a t i o n (C) of BSA i s given by:
M
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=
vp + VI + vs
where M i s t h e mass of p r o t e i n added, and t h e denominat o r i s t h e t o t a l volume a v a i l a b l e t o p r o t e i n [ t h e sum o f t h e pore volume i n t h e g e l p a r t i c l e s (V,),
the inter-
s t i t i a l volume between t h e p a r t i c l e s i n t h e packed g e l l a y e r (V,)
, and
The value o f (Vp
t h e s u p e r n a t a n t volume (Vs = 0.5 m l ) 1.
+ V,)
was determined t o b e 0.32 m l i n
0.5 m l of packed g e l .
The t o t a l volume o f l i q u i d , excluding t h e pore vo 1i n a suspension i d e n t i c a l t o t h e above was
ume (V,),
measured by u s i n g b a k e r ' s y e a s t i n s t e a d of BSA.
The
diameter of y e a s t i s approximately 5 um, and t h e organ-
isms cannot p e n e t r a t e t h e g e l pores, which have a s i z e l i m i t of approximately 0.02 s o n a l communication). pension gel.
(lo7
u m (P.L.
Biochemicals, per-
An a l i q u o t ( 0 . 3 m l ) of y e a s t sus-
cells/ml) w a s added t o 0.5 m l o f packed
A f t e r t h e t o t a l volume w a s a d j u s t e d t o one m l ,
t h e g e l was stirred f o r 15 minutes and c e n t r i f u g e d a t low speed.
The y e a s t c o n c e n t r a t i o n i n t h e s u p e r n a t a n t
w a s then determined with a hemocytometer, and Vs w a s c a l c u l a t e d t o be 0.63 ml.
+ VI
Therefore, VI was 0.13
m l (26% o f t h e packed g e l volume) and Vp w a s 0.19 m l
487
ACETATE KINASE ( 3 8 % o f t h e packed g e l volume).
T h i s v a l u e o f Vp was
used t o c a l c u l a t e t h e c o n c e n t r a t i o n [ E L I o f enzymel i g a n d complex i n t h e p o r e s o f t h e g e l . RESULTS
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P u r l f i c a t i on o f Acetate K i n a s e The p a r t i a l l y p u r i f i e d commercial acetate k i n a s e p r e p a r a t i o n was a d s o r b e d by g e l t y p e s 2 and 3 , i n which AMP i s bound t h r o u g h t h e a d e n i n e r i n g , b u t n o t by g e l
i n which AMP i s bound t h r o u g h t h e r i b o s e group.
type 4 ,
These o b s e r v a t i o n s s u g g e s t t h a t t h e s p e c i f i c i t y of t h e enzyme t o w a r d s a d e n i n e n u c l e o t i d e s is n o t m a n i f e s t e d t h r o u g h t h e a d e n i n e moiety.
The a b i l i t y of acetate k i n -
a s e t o u s e guanosine 5 ' - t r i p h o s p h a t e 5'-triphosphate action'
'
(GTP) and i n o s i n e
( I T P ) as s u b s t r a t e s i n t h e r e v e r s e re-
is i n accordance w i t h t h i s i n t e r p r e t a t i o n .
G e l type 3 exhibited a s t r o n g e r a f f i n i t y €or acetate
k i n a s e t h a n d i d g e l t y p e 2 and w a s u s e d i n a l l subseq u e n t experiments. F i g . 1 shows t h e s p e c i f i c a d s o r p t i o n and e l u t i o n from t y p e 3 g e l o f t h e p a r t i a l l y p u r i f i e d acetate k i n a s e from Sigma.
When BSA w a s added t o t h e c o n t a c t -
i n g s o l u t i o n , v i r t u a l l y a l l o f i t passed through t h e column, w h e r e a s acetate k i n a s e w a s r e t a i n e d and w a s e l u t e d by 1 0 mM ADP b u t n o t by an N a C l s o l u t i o n o f comparable i o n i c s t r e n g t h .
The p r e s e n c e o f Mg2+ and
a low i o n i c s t r e n g t h w a s b e n e f i c i a l f o r b i n d i n g t h e
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1 2 0 1 ,
0
I
10
,
I
,
,
I
20 30 EFFLUENT VOLUME ( m l )
,6
40
FIGURE 1
A f f i n i t y chromatography of p a r t i a l l y p u r i f i e d acet a t e k i n a s e (Sigma) on a n agarose-bound C 8 - (6-aminohexy1)-AMP g e l . The column w a s 6 x 0 . 9 c m , and t h e A. E i g h t e e n m l o f soluflow r a t e was 0 . 4 ml/minute. t i o n c o n t a i n i n g a c e t a t e k i n a s e ( 2 2 U / m l ) , 1 0 mM MgC12, 1 mM DTT, and 4 mg/ml BSA i n 0 . 1 M phosphate b u f f e r a t pH 7.4 were a p p l i e d t o t h e column a t room t e m p e r a t u r e . B. Wash s o l u t i o n : 1 mM DTT and 1 0 mM MgC12 a t pH 7.4. C. E l u t i n g s o l u t i o n : same a s (B) b u t w i t h 1 0 mM ADP. S p e c i f i c a c t i v i t y of t h e enzyme p r i o r t o a d d i t i o n of BSA w a s 930 U/mg; a f t e r a d d i t i o n o f BSA i t w a s 5.5 U/mg. Of t h e 4 0 0 U o f enzyme added t o t h e column, 222 U were bound and recovered on e l u t i o n . S p e c i f i c a c t i v i t y of t h e recovered enzyme was 6 0 0 0 U/mg p r o t e i n .
enzyme t o t h e g e l .
The s p e c i f i c a c t i v i t y of t h e p u r i -
f i e d enzyme w a s 6 , 0 0 0 U p e r mg p r o t e i n , which r e p r e s e n t s a s i x - f o l d p u r i f i c a t i o n over t h e DCT-activated enzyme a p p l i e d t o t h e column ( s p e c i f i c a c t i v i t y 930 U p e r mg p r o t e i n ) .
The p u r i f i c a t i o n o f acetate k i n a s e t o
such a h i g h d e g r e e h a s n o t been r e p o r t e d p r e v i o u s l y . The h i g h e s t s p e c i f i c a c t i v i t i e s i n t h e l i t e r a t u r e r a n g e from 1 6 0 t o 300 U p e r mg p r o t e i n .
I
Polyacrylamide
g e l e l e c t r o p h o r e s i s of t h e p u r i f i e d enzyme i s o l a t e d from t h e Sigma p r e p a r a t i o n r e v e a l e d o n l y a s i n g l e band.
489
ACETATE KINASE
The absence o f DTT d u r i n g e l e c t r o p h o r e s i s , however, r e s u l t e d i n one l a r g e d i f f u s e band.
T h i s phenomenon
p r o b a b l y a r i s e s from t h e p r e s e n c e of b o t h reduced and o x i d i z e d forms o f t h e enzyme.
Assays o f g e l s e c t i o n s
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soaked i n DTT s o l u t i o n showed enzyme a c t i v i t y througho u t t h e s t a i n e d zone. The a d s o r p t i o n - e l u t i o n p r o f i l e o f t h e c r u d e , c e l l f r e e e x t r a c t prepared from E . c o l i u s i n g t y p e 3 g e l i s shown i n Fig.
2.
Adenylate k i n a s e , which i n t e r f e r e s i n
t h e acetate k i n a s e a s s a y system, showed no a f f i n i t y f o r the gel.
The s p e c i f i c a c t i v i t y of t h e acetate k i n a s e
EFFLUENT VOLUME Im l 1
FIGURE 2 A f f i n i t y chromatography o f a c r u d e e x t r a c t o f E. The column of F i g . 1 w a s used, b u t t h e f l o w r a t e was 0 . 1 7 ml/minute. A. Nineteen m l o f c r u d e e x t r a c t i n 0 . 1 M KH2P04, 1 mM DTT, and 1 0 mM MgC12 were a p p l i e d . E n t e r i n g enzyme c o n c e n t r a t i o n s were 2 . 2 U / m l f o r adenyla t e k i n a s e and 3 2 . 3 U / m l f o r acetate k i n a s e ( s p e c i f i c a c t i v i t y 1 4 U/mg p r o t e i n ) . B. Wash s o l u t i o n : 1 mM DTT and 1 mM MgC12. C. E l u t i o n by 1 mM DTT, 1 mM MgC12, and 1 0 mM ADP. O f t h e 6 1 4 U of a c e t a t e k i n a s e a p p l i e d , 3 6 8 were r e t a i n e d by t h e column, and 3 3 5 U ( 9 1 % o f t h o s e bound) were r e c o v e r e d a t a s p e c i f i c a c t i v i t y of 5 3 0 U/mg.
coli.
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490
e l u t e d w i t h 1 0 mM ADP w a s 530 U p e r mg p r o t e i n , which r e p r e s e n t s a 38-fold p u r i f i c a t i o n from t h e crude extract.
Although d i s c g e l e l e c t r o p h o r e s i s r e v e a l e d
s e v e r a l p r o t e i n bands , t h i s p r e p a r a t i o n c o n t a i n e d no
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p r o t e a s e , phosphatase, o r a d e n y l a t e k i n a s e a c t i v i t i e s and produced t h e same e q u i l i b r i u m n u c l e o t i d e concentrat i o n s a s d i d t h e acetate k i n a s e o b t a i n e d from Sigma, thereby i n d i c a t i n g t h e absence o f s i g n i f i c a n t A T P a s e activity. The enzyme a c t i v a t i o n observed upon t h e a d d i t i o n of DTT i s c o n s i s t e n t w i t h t h e e x i s t e n c e o f a c t i v e and
i n a c t i v e forms o f t h e enzyme.
To d e t e r m i n e whether t h e
i n a c t i v e form can b i n d t o t h e column, a p a r t i a l l y reacti v a t e d enzyme s o l u t i o n was a p p l i e d u n t i l t h e column approached s a t u r a t i o n .
The enzyme s o l u t i o n n o t r e t a i n -
e d by t h e g e l w a s c o l l e c t e d , and t h e column w a s t h e n e l u t e d w i t h 1 0 mM ADP t o form a second s o l u t i o n . s o l u t i o n s were e q u i l i b r a t e d w i t h 1 mM DTT.
Both
After 24
h o u r s , t h e a c t i v i t y o f t h e enzyme n o t r e t a i n e d had doubled, while t h a t o f t h e enzyme bound and t h e n e l u t e d had n o t i n c r e a s e d .
These r e s u l t s s u g g e s t t h a t o n l y
c a t a l y t i c a l l y a c t i v e enzyme i s r e t a i n e d by t h e i m b i l i z e d AMP.
E q u i l i b r i u m Binding S t u d i e s The a d s o r p t i o n p r o c e s s may be expressed by: E + L C E L
(2)
where E is t h e f r e e enzyme i n s o l u t i o n , L i s t h e l i g a n d
ACETATE KINASE
491
(AMP) a t t a c h e d t o t h e a f f i n i t y g e l , and EL i s t h e imb i l i z e d enzyme-ligand complex.
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tion constant
(%)
An e q u i l i b r i u m a s s o c i a -
can be d e f i n e d a s :
Adsorption i s o t h e r m s measured w i t h t h e Sigma ace-
t a t e k i n a s e p r e p a r a t i o n and t h e a f f i n i t y g e l i n column and s u s p e n s i o n experiments a r e shown i n Fig.
3.
Ident-
i c a l l i n e a r i s o t h e r m s d e s c r i b e d t h e amount of enzyme adsorbed d u r i n g b o t h column and s u s p e n s i o n e x p e r i m e n t s , and t h e s l o p e of t h e l i n e gave t h e v a l u e o f t h e p r o d u c t K [ L ] , which i s a measure o f t h e enzyme b i n d i n g capaL
c i t y of t h e gel.
Although b o t h enzyme p r e p a r a t i o n s
were a p p l i e d t o t h e column w i t h s o l u t i o n s o f t h e same composition, t h e s l o p e f o r t h e Sigma p r e p a r a t i o n w a s 8.5 and t h a t f o r t h e E. c o l i e x t r a c t 5.6.
This d i f f e r -
e n c e may b e due t o d i f f e r e n t l e v e l s of i m p u r i t i e s i n t h e two p r e p a r a t i o n s .
Column s i z e and geometry, f l o w
r a t e , r e s i d e n c e t i m e , t o t a l armunt of enzyme c o n t a c t e d , and t h e enzyme c o n c e n t r a t i o n i n the e n t e r i n g s o l u t i o n
w e r e varied.
The p r o d u c t K L [ L ] c o r r e l a t e d o n l y w i t h
t h e c o n c e n t r a t i o n o f enzyme s o l u t i o n a p p l i e d t o t h e column. The l i n e a r i t y of t h e p l o t s i n d i c a t e s t h a t K L I L l
w a s c o n s t a n t , and s u g g e s t s t h a t t h e e f f e c t i v e l i g a n d c o n c e n t r a t i o n was n o t s i g n i f i c a n t l y reduced by t h e
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492
FIGURE 3 Adsorption isotherms obtained from column purifications and equilibrium experiments with gel suspensions in 0.1 M KH2P04, 10 mM Mg++, and 1 mM DTT. Acetate kinase (Sigma): 0 , gel suspension experiment, 0 , column experiment; crude E. coli extract: A, gel suspension experiment, A , column experiment. In the gel suspension experiments, [El is the equilibrium concentration of enzyme in the supernatant, and [EL] is the quantity of enzyme adsorbed divided by the calculated gel pore volume; in the column purification experiments, [El is the enzyme concentration in the inlet stream, and [EL] is calculated from the amount of enzyme retained on the column.
ACETATE KINASE
493
enzyme a d s o r p t i o n .
T h i s f i n d i n g i s s u p p o r t e d by an est-
imated maximum g e l c a p a c i t y of 4.2
x lo6 U p e r m i of
pore volume ( c a l c u l a t e d from 5.8 p m o l i m m b i l i z e d AMP p e r m l of packed g e l ; m o l e c u l a r weight 4 1 , 0 0 0 f o r ace-
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t a t e k i n a s e from E . c o l i 1 2 ; 6,000 U/mg f o r t h e p u r i f i e d p r o t e i n ; one enzyme bound t o each A M P ) , which i s approximately
lo5
t i m e s as g r e a t as t h e h i g h e s t c o n c e n t r a t i o n
of enzyme used.
Thus, even i f o n l y a v e r y s m a l l f r a c -
t i o n of t h e AMP w e r e a c c e s s i b l e , i t would s t i l l be i n g r e a t excess compared t o t h e q u a n t i t i e s of enzyme adsorbed. E f f e c t s o f Magnesium I o n C o n c e n t r a t i o n and Ionic Strength E a r l y experiments s u g g e s t e d t h a t magnesium i o n conc e n t r a t i o n and i o n i c s t r e n g t h e x e r t major i n f l u e n c e s on g e l c a p a c i t y .
The e f f e c t s o f b o t h w e r e t h e r e f o r e
e x p l o r e d i n an a t t e m p t t o m a x i m i z e K L [ L ] .
Fig. 4 p r e -
s e n t s v a l u e s o f K L [ L ] as a f u n c t i o n o f i o n i c s t r e n g t h a t two c o n c e n t r a t i o n s of M g C 1 2 .
Data a t t h e two high-
e s t i o n i c s t r e n g t h s were t a k e n from g e l s u s p e n s i o n experiments.
The o t h e r d a t a were o b t a i n e d from t h e
column wash s t e p o f p u r i f i c a t i o n s o f t h e Sigma enzyme preparation.
++
Wash s o l u t i o n s c o n t a i n i n g t h e same Mg
c o n c e n t r a t i o n b u t d i f f e r i n g i n N a C l c o n c e n t r a t i o n s were a p p l i e d t o a column c o n t a i n i n g a d s o r b e d enzyme u n t i l t h e enzyme c o n c e n t r a t i o n o f t h e e f f l u e n t r e a c h e d a c o n s t a n t v a l u e , which w a s used t o estimate K L [ L ] .
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0
60
120 180 240 I O N I C STRENGTH I m M )
300
FIGURE 4 KL[L] as a f u n c t i o n o f i o n i c s t r e n g t h . Except f o r t h e d a t a of t h e t w o highest i o n i c s t r e n g t h s , values of K L [ L ] were o b t a i n e d d u r i n g column p u r i f i c a t i o n s by d e t e r m i n i n g t h e enzyme c o n c e n t r a t i o n i n t h e wash s o l u The t i o n as i t s c o n c e n t r a t i o n of N a C l was i n c r e a s e d . s o l u t i o n s a s a p p l i e d c o n t a i n e d o n l y NaC1, 1 mM DTT, a n d t h e c o n c e n t r a t i o n o f MgC12 i n d i c a t e d (0, 6 0 mM; 0 , 1 0 mM) KL [ L ] was computed w i t h [ E l a s t h e a c e t a t e k i n a s e c o n c e n t r a t i o n i n t h e e f f l u e n t a t s t e a d y s t a t e and [EL] a s t h e c a l c u l a t e d c o n c e n t r a t i o n o f i m m o b i l i z e d enzyme.
.
S i n c e enzyme c o n c e n t r a t i o n s i n t h e e f f l u e n t w e r e n o t s e n s i t i v e t o l i q u i d f l o w rates, w e c o n c l u d e d t h a t t h e enzyme c o n c e n t r a t i o n s were c o n t r o l l e d b y t h e enzymeligand equilibrium.
A s l i g h t increase i n t h e ionic
s t r e n g t h of t h e s o l u t i o n c a u s e d a l a r g e decrease i n t h e measured v a l u e of K L I L I a t b o t h c o n c e n t r a t i o n s of MgC12. The s l o p e s of t h e l i n e s i n Fig. a v e r a g e v a l u e of -10.9.
4 are s i m i l a r , w i t h a n
The v a r i a t i o n i n K L I L l w i t h
i o n i c s t r e n g t h may be e x p r e s s e d by: A ( l 0 g K L I L I ) = -10.9Ap
(4)
where p i s t h e i o n i c s t r e n g t h . KL[L] w a s n e x t measured w i t h t h e Sigma a c e t a t e k i n -
ase p r e p a r a t i o n a t i n c r e a s i n g c o n c e n t r a t i o n s of MgC12.
49 5
ACETATE KINASE
Because o f t h e a p p r e c i a b l e c o n t r i b u t i o n by MgCl, t o t h e i o n i c s t r e n g t h , no a t t e m p t w a s made t o k e e p i o n i c s t r e n g t h constant.
To a v o i d f o r m a t i o n o f magnesium
p h o s p h a t e p r e c i p i t a t e s , measurements were made w i t h
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enzyme-gel buffer.
suspensions with T r i s r a t h e r than phosphate
A s shown i n F i g .
5 , KLILI i n c r e a s e d r a p i d l y
FIGURE 5 Dependence o f KL [ L l on MgC12. V a l u e s were o b t a i n e d from e q u i l i brium experiments w i t h Sigma a c e t a t e k i n a s e and a f f i n i t y g e l s u s p e n s i o n s i n 1 mM DTT (0, i n 0 . 1 M T r i s b u f f e r ; 0 , i n 0.01 M T r i s ) . The maximum i n K L [ L ] o c c u r r e d a t [MgC12 I o f 60 mM w i t h 0 . 0 1 M T r i s and of 4 0 mM w i t h 4 0 mM T r i s .
496
SWARTZ ET AL.
w i t h i n c r e a s i n g c o n c e n t r a t i o n s of M g C 1 2 , r e a c h e d a peak, and t h e n d e c l i n e d . Equation 4 w a s a p p l i e d t o t h e r e s u l t s i n Fig. 5 t o s e p a r a t e t h e e f f e c t s r e s u l t i n g from c h a n g e s i n i o n i c
++ I .
The con-
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s t r e n g t h from t h o s e due t o changes i n [Mg
d i t i o n s c o r r e s p o n d i n g t o t h e maximum v a l u e i n Fig. 5 (KL[L] = 58,
[MgC12] = 6 0 mM,
p = 263 mM) were s e l e c t e d
as a b a s i s €or comparison, and Ap f o r e a c h d a t a p o i n t was t a k e n t o b e t h e d i f f e r e n c e between t h e a c t u a l i o n i c s t r e n g t h and 0.268 M.
The v a l u e o f A(1og K L I L I ) w a s
t h e n c a l c u l a t e d from E q u a t i o n 4 and added t o t h e logarithm of t h e KL[L] v a l u e a c t u a l l y measured.
Fig.
6 is
++ I
a p l o t of t h e c a l c u l a t e d v a l u e of K L I L I v e r s u s [Mg
t h a t would have been o b t a i n e d i f i t were p o s s i b l e t o m a i n t a i n t h e i o n i c s t r e n g t h c o n s t a n t a t 0.268 out.
M through-
The two s e p a r a t e c u r v e s o b t a i n e d i n c r e a s e d mono-
t o n i c a l l y w i t h [MgC12] and c o r r e s p o n d e d t o t h e two c o n c e n t r a t i o n s o f T r i s b u f f e r used. To show t h a t t h e r e s u l t s o f t h e enzyme-gel suspens i o n c o n t a c t s i n Fig.
5 a l s o a p p l i e d t o column p u r i f i c a -
t i o n s , a n experiment w a s performed w i t h t h e Sigma enzyme under o p t i m a l c o n d i t i o n s f o r a d s o r p t i o n . f i v e m l of enzyme s o l u t i o n ( 5 . 3 9 U / m l )
Sixty-
i n 60 mM MgC12,
26 mM (NH4)2S04, 3 mM DTT, and 0 . 0 1 M T r i s a t pH 7.4 ( p = 0.263 M ) were a p p l i e d t o a column c o n t a i n i n g 2 m l
of g e l (Vp = 0.76 m l ) .
Of t h e 350 U a p p l i e d , 240 w e r e
ACETATE KINASE
49 7
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'o*ooo*
0
20
40 [MpCI,]
60 80 (mM)
100
FIGURE 6 The e f f e c t o f [MgCl21 on K L [ L ] when c o r r e c t e d f o r t h e e f f e c t of i o n i c s t r e n g t h . Equation 4 was used t o a d j u s t measured values of KL[LI t o t h o s e t h a t would b e e x p e c t e d i f p had been c o n s t a n t a t 0 . 2 6 8 M. 0, i n 0.1 M T r i s buffer; 0 , i n 0.01 M T r i s .
r e t a i n e d by t h e g e l .
T h i s compares w e l l w i t h t h e capa-
c i t y e x p e c t e d on t h e b a s i s o f t h e g e l s u s p e n s i o n e x p e r i ments ( K L [ L ] [ E ] V p = 238 U).
When t h e column w a s e l u t e d
w i t h 10 m l of a s o l u t i o n c o n t a i n i n g 1 0 mM ADP, 6 0 mM MgC12, and 1 mM DTT, a d j u s t e d t o pH 7.4, 205
u
(85%)
were r e c o v e r e d a t a n a v e r a g e s p e c i f i c a c t i v i t y of 2155 U p e r mg p r o t e i n .
These r e s u l t s r e p r e s e n t a s i g n i f i -
c a n t improvement i n performance i n comparison t o t h o s e o f F i g . 1, wherein 222 U were r e t a i n e d by 4 r n l o f
SWART2 ET AL.
49 8
g e l a t a n e n t e r i n g enzyme c o n c e n t r a t i o n of 2 2 U/ml.
If
t h e experiment of F i g . 1 had been conducted w i t h a g e l volume of 2 m l and an e n t e r i n g enzyme c o n c e n t r a t i o n of 5.39 U / m l ,
o n l y 27 U would have been e x t r a c t e d from t h e
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entering solution.
Although t h e p r o d u c t o f t h e f i n a l
experiment w a s n o t a s p u r e as t h a t o b t a i n e d i n t h e experiment shown i n F i g . 1 (2155 vs. 6 0 0 0 U/mg p r o t e i n ) , t h e column c a p a c i t y h a s been i n c r e a s e d n i n e - f o l d .
This
i n c r e a s e may b e a t t r i b u t e d t o t h e i n c r e a s e i n [MgCl21 from 1 0 t o 60 mM and t h e d e c r e a s e i n i o n i c s t r e n g t h from 331 t o 263 mM. D I S CUSSION
W e have shown t h a t AMP, bound t h r o u g h i t s a d e n i n e moiety t o a g a r o s e , a c t s as an e f f e c t i v e l i g a n d f o r t h e b i o s p e c i f i c a d s o r p t i o n o f acetate k i n a s e .
Partially
p u r i f i e d acetate k i n a s e o b t a i n e d from Sigma was p u r i f i e d t o a p p a r e n t homogeneity by t h i s method.
By a s i n -
g l e p a s s t h r o u g h t h e a f f i n i t y column, a c e l l - f r e e ex-
t r a c t from E. c o l i was p u r i f i e d t o a d e g r e e s u i t a b l e f o r u s e i n a l a r g e - s c a l e enzyme r e a c t o r ( a b s e n c e of p r o t e a s e , p h o s p h a t a s e and A T P a s e a c t i v i t i e s )
.
The a d s o r p t i o n i s o t h e r m s were l i n e a r w i t h b o t h t h e Sigma enzyme p r e p a r a t i o n and t h e c r u d e E . c o l i e x t r a c t , and y i e l d e d KLILI v a l u e s of 9.4 and 6.3, i n 0 . 1 M phosphate (pH 7 . 4 )
respectively,
and 1 0 mM Mg++.
The d i f f e r -
ence i n s l o p e s may be a t t r i b u t e d t o t h e h i g h e r level of Contaminant p r o t e i n s i n t h e crude e x t r a c t , which would
499
ACETATE KINASE
i n c r e a s e t h e i o n i c s t r e n g t h o f t h e s o l u t i o n , r e d u c e by
++
c h e l a t i o n t h e e f f e c t i v e Mg
c o n c e n t r a t i o n , and p o s s i -
b l y compete f o r t h e l i g a n d . Both t h e i o n i c s t r e n g t h o f t h e s o l u t i o n and t h e
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m a gne s ium i o n concen tr a t i on s t r o n q l y in f 1uen ced acetate k i n a s e adsorption.
The v e r y pronounced i n c r e a s e i n
KL ILI w i t h d e c r e a s i n g i o n i c s t r e n g t h w a s unexpected,
b u t t h e r e s u l t s a r e c o n s i s t e n t w i t h r o u t i n e l y observed column performance, t h a t i s , when t h e columns were washed w i t h s o l u t i o n s of a lower i o n i c s t r e n g t h t h a n t h a t o f t h e enzyme s o l u t i o n s a p p l i e d , v e r y l i t t l e loss o f enzyme from t h e column was observed.
I f t h e lower
i o n i c s t r e n g t h had n o t caused a d r a m a t i c i n c r e a s e i n t h e e q u i l i b r i u m c o n s t a n t f o r a d s o r p t i o n , and K L [ L ] had remained t h e same, t h e n t h e e x i t i n g enzyme c o n c e n t r a t i o n d u r i n g t h e wash s t e p f o l l o w i n g s a t u r a t i o n o f t h e column would have been e q u a l t o t h e c o n c e n t r a t i o n o f t h e enzyme s o l u t i o n o r i g i n a l l y a p p l i e d .
The i n s i g n i f i -
c a n t l o s s of enzyme r o u t i n e l y observed d u r i n g t h e wash s t e p o f column p u r i f i c a t i o n s under t h e s e c o n d i t i o n s i s p o s s i b l e o n l y w i t h v e r y h i g h v a l u e s of K L I L I .
The
dependence o f KLILI on i o n i c s t r e n g t h is a l s o i n q u a l i t a t i v e agreement w i t h t h e o b s e r v a t i o n s of Robinson
a1.,13
et
who found t h a t t h e b i n d i n g of 6 - g a l a c t o s i d a s e
t o an a f f i n i t y matrix decreased s u b s t a n t i a l l y w i t h i n c r e a s i n g i o n i c s t r e n g t h of t h e b u f f e r .
SWART2 ET AL.
500
The s t r o n g dependence of t h e g e l c a p a c i t y on
++ [Mg ]
c a n n o t b e e x p l a i n e d s i m p l y by t h e i n c r e a s e d
++ 3 .
MgAMP c o n c e n t r a t i o n s w i t h i n c r e a s i n g [ M g
The i n -
crease i n [MgAMP] i s a t most a f i r s t o r d e r f u n c t i o n o f
++ 1, ++ [Mg I
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[Mg
whereas KL [L] i n c r e a s e s l o g a r i t h m i c a l l y w i t h i n Fig. 6.
t o i n c r e a s e KL.
Mg++ may a c t d i r e c t l y on t h e enzyme I t i s a l s o p o s s i b l e t h a t h i g h local
c o n c e n t r a t i o n s o f u n c h e l a t e d AMP (which e x i s t s p r e d o w i n a n t l y a s AMP2' charged g e l .
a t pH 7 . 4 ) would produce a n e g a t i v e l y
I t h a s been shown t h a t i n s o l u t i o n s o f
low i o n i c s t r e n g t h , t h e pH i n t h e microenvironment n e a r t h e g e l can b e s u b s t a n t i a l l y lower t h a n t h a t o f the bulk solution.14
++ 1,
The e f f e c t o f i n c r e a s i n g [Mg
and t h u s [MgAMP], i s t o d e c r e a s e t h e l o c a l n e g a t i v e charge, t h e r e b y i n c r e a s i n g t h e pH i n t h e microenvironment o f t h e l i g a n d and p e r h a p s c h a n g i n g enzyme a f f i n i t y f o r t h e ligand.15
Our r e s u l t s have i m p o r t a n t i m p l i c a t i o n s f o r t h e o p e r a t i o n and s c a l e - u p of enzyme p u r i f i c a t i o n p r o c e s s e s u t i l i z i n g b i o s p e c i f i c adsorption.
A l l three steps
(enzyme a d s o r p t i o n , removal of c o n t a m i n a n t s , and enzyme e l u t i o n ) are a f f e c t e d by t h e i n t e r a c t i o n between enzyme and l i g a n d .
A l a r g e r g e l c a p a c i t y may be o b t a i n e d by
i n c r e a s i n g KL [L] and/or
[El
.
Concentration o f t h e
c r u d e enzyme s o l u t i o n may t h u s be advantageous i f i o n i c s t r e n g t h i s n o t s i g n i f i c a n t l y a f f e c t e d and i f v i s c o s i t y
ACETATE KINASE
501
i s n o t i n c r e a s e d s u c h t h a t f l o w t h r o u g h t h e column becomes d i f f i c u l t .
The v a l u e o f K L [ L ] may b e i n c r e a s e d
++
s e v e r a l - f o l d by a d j u s t i n g i o n i c s t r e n g t h and Mg However, as i n d i c a t e d by Robinson
centration.
con-
% &.13
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and by o u r d a t a , p r o d u c t p u r i t y may d e c r e a s e a s i o n i c s t r e n g t h o f t h e c r u d e s o l u t i o n i s d e c r e a s e d and more non-specific
a d s o r p t i o n occurs. ACKNOWLEDGEMENT
T h i s s t u d y w a s s u p p o r t e d by Grant No. GI34284 from t h e N a t i o n a l S c i e n c e Foundation. REFERENCES AND NOTES 'Present address:
T a t e and L y l e Ltd.,
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