enzyme electrode for fructose, glucose-6-phosphate and atp determination

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Enzyme Electrode for Fructose,Glucose-6-Phosphate and ATPDeterminationF. Schubert a , D. Kirstein a , F. Scheller a , M.Abraham b & L. Boross ba Central Institute of- Molecular Biology, Academyof Sciences of the CDR, Department of AppliedEnzymology, 1115, Berlin-Buch, GDRb Attila Jozsef University Institute of Biochemistry,Szeged, H-6726, Szeged, HungaryVersion of record first published: 03 Jan 2007.

To cite this article: F. Schubert , D. Kirstein , F. Scheller , M. Abraham & L. Boross(1986): Enzyme Electrode for Fructose, Glucose-6-Phosphate and ATP Determination,Analytical Letters, 19:21-22, 2155-2167

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ANALYTICAL LETTERS, 1 9 ( 2 1 & 2 2 ) , 2 1 5 5 - 2 1 6 7 ( 1 9 8 6 )

ENZYME ELECTRODE FOR FRUCTOSE, GLUCOSE-6-PHOSPHATE

AND ATP DETERMINATION

F . Schuber t , D . K i r s t e i n and F . S c h e l l e r

C e n t r a l I n s t i t u t e of. M o l e c u l a r B i o l o g y , Academy o f Sc iences o f t h e CDR

Department o f A p p l i e d Enzymology 1115 Ber l i n -Buch , GDR

M. Abraham and L. Boross

A t t i l a Jozse f U n i v e r s i t y I n s t i t u t e o f B i o c h e m i s t r y , Szeged,

H-6726 Szeged, Hungary

Keywords: F r u c t o s e sensor , Bienzyme e l e c t r o d e ,

S u b s t r a t e c o m p e t i t i o n

ABSTRACT

A bienzyme e l e c t r o d e f o r g lucose-6-phosphate and ATP

based on the s e q u e n t i a l r e a c t i o n s o f co immob i l i zed

g lucose-6-phosphate dehydrogenase and hexok inase and f o r

f r u c t o s e u s i n g t h e c o m p e t i t i o n w i t h g lucose has been deve-

loped. E l e c t r o c h e m i c a l i n d i c a t i o n i s per fo rmed u s i n g a i r -

o x i d a t i o n o f reduced N-methy lphenazin ium i o n . The sensor

responds l i n e a r l y t o f r u c t o s e up t o 3 mM w i t h a l ower

d f t e c t i o n l i m i t o f 0.3 mhl.

Copyright 0 1986 by Marcel Dekker, Inc

2 1 5 5

0003-27 19/86/ 192 1-21 SS$3.50/0

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2156 SCHUBERT ET A L ,

INTRODUCTION

Enzymatic methods f o r f r u c t o s e d e t e r m i n a t i o n use

e i t h e r f r u c t o s e dehydrogenase' o r t h e s e q u e n t i a l a c t i o n

o f hexokinase, phosphoglucose isomerase and glucose-6-

phosphate dehydrogenase' f o l l o w e d by spec t ropho tomet r i c

measurement o f reduced e l e c t r o n acceptors . Furthermore,

a b iosensor-based method has been descr ibed3 c o n s i s t i n g

o f chemical f r u c t o s e i s o m e r i z a t i o n t o g lucose w i t h sub-

sequent g lucose measurement by a g lucose ox idase enzyme

e l e c t r o d e . Owing t o t h e i r i n h e r e n t enzyme consumption

t h e former procedures are expensive. Though a v o i d i n g

t h i s drawback, t h e procedure u s i n g an enzyme e l e c t r o d e

s u f f e r s from t h e l abo r iousness o f chemical i somer i za -

t i o n r e q u i r i n g i n c u b a t i o n a t 75 C and pH 14. Accor-

d i n g t o p r o d u c t i n f o r m a t i o n s , f r u c t o s e measurement i s

a l s o p o s s i b l e w i t h t h e Ye l low Spr ings I n d u s t r i a l Ana-

l y z e r , u s i n g a ga lac tose ox idase membrane. However, i n

v iew o f r e c e n t l y p u b l i s h e d r e s u l t s concern ing t h e spe-

c i f i t y o f ga lac tose ox idase4

t o be ques t i onab le .

0

t h i s p o s s i b i l i t y seems

The p resen t paper r e p o r t s t h e a p p l i c a t i o n o f a b i -

enzyme e l e c t r o d e u s i n g hexokinase and glucose-6-

phosphate dehydrogenase f o r f r u c t o s e de te rm ina t ion .

The method i s based on t h e c o m p e t i t i o n o f f r u c t o s e and

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ENZYME ELECTRODE 2 1 5 7

g l u c o s e f o r h e x o k i n a s e . E l e c t r o c h e m i c a l s e n s i n g is p e r -

fo rmed v i a t h e c h e m i c a l m e d i a t o r , N-methy lphenaz in ium

i o n . The NAOPH p r o d u c e d i n t h e g lucose-6-phosphate de-

hydrogenase r e a c t i o n i s r e o x i d i z e d by t h e N-methy lphena-

z i n i u n i i o n and t h e r e d u c e d m e d i a t o r i s r e o x i d z e d by mole-

c u l a r oxygen. The change i n oxygen c o n c e n t r a t i o n i s

measured by a C l a r k oxygen e l e c t r o d e . The r e a c t i o n s t e p s

i n v o l v e d i n t h e enzyme e l e c t r o d e a l s o p e r m i t measurement

o f g lucose-6-phosphate and ATP.

EXPERIMENTAL

hl a t e r i a 1 s

Y e a s t h e x o k i n a s e ( E C 2.7.1.1) was o b t a i n e d f r o m

Merck , D a r m s t a d t , g lucose-6-phosphate dehydrogenase

(GGP-DH, E C 1.1.1.49) and g lucose-6-phosphate ( G 6 P ) were

f r o m B o e h r i n g e r Mannheim. ATP and NADP+ were p u r c h a s e d

f r o m Reana l , Budapest . N-methy lphenaz in ium i o n as t h e

m e t h y l s u l f a t e s a l t (NMP+) was f r o m Chemapol, Prague.

A l l o t h e r r e a g e n t s were a n a l y t i c a l r e a g e n t g r a d e and a l l

t h e s o l u t i o n s were p r e p a r e d i n d i s t i l l e d , d e i o n i z e d

w a t e r . The s t a n d a r d measur ing b u f f e r was 0.1 M Tr is -HC1

b u f f e r , pH 7.0, c o n t a i n i n g 10 mM MgC12. B e f o r e t h e

measurements, NMP' was added t o g i v e 1 mM f i n a l concen-

t r a t i o n .

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2158 SCHUBERT ET A L

Enzyme E l e c t r o d e P r e p a r a t i o n

Bienzyme membranes were p r e p a r e d by g e l a t i n en-

5 t r a p m e n t o f t h e enzymes as d e s c r i b e d p r e v i o u s l y . 7 U ( 5 / u l ) o f h e x o k i n a s e and 5 . 8 U ( l O / u l ) o f G6P-DI-1

n

were a p p l i e d p e r cmL o f nienibraric. Membranes were s t o r e d

under r e f r i g e r a t i o n u n t i l use. A p i e c e o f a p p r o x i m a t e l y

5 x 5 mm o f t h e membrane was used f o r sensor p r e p a r a -

t i o n . I t was sandwiched between a p o l y p r o p y l e n e membrane

and a d i a l y s i s membrane (VEB CK B i t t e r f e l d , d = 15 um)

and t h i s ar rangement was f i x e d t o t h e t i p o f an oxygen

e l e c t r o d e ( P t , Ag/AgCl, R a d e l k i s , Budapest) w i t h an 0-

r i n g and a p l a s t i c cap.

/

A p p a r a t u s and P r o c e d u r e

The enzyme sensor was c o n n e c t e d t o t h e R a d e l k i s

G lucose A d a p t e r ( R a d e l k i s , Budapest) combined w i t h a

r e c o r d e r . Measurements were c a r r i e d o u t i n 5 m l o f

s t i r r e d background b u f f e r a t 2 8 2 0.5'. A f t e r ad-

d i t i o n o f c o f a c t o r s and e q u i l i b r a t i o n samples o f

50 u l were i n j e c t e d . The s t e a d y s t a t e c u r r e n t changes

were r e c o r d e d . /

RESULTS AND DISCUSSION

b

The r e a c t i o n s t a k i n g p l a c e i n t h e b ienzyme membrane

a r e shown i n F i g . 1. G6P fo rmed f r o m g l u c o s e by hexo-

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ENZYME ELECTRODE 2159

NADP+ glucose fructose FIG. 1 . Principle of the bienzyme sensor

k i n a s e i s o x i d i z e d by GGP-DH w i t h concomi tan t NADPH

f o rma t ion . The reduced p y r i d i n e n u c l e o t i d e i s measured

v i a i t s r e a c t i o n w i t h [\IMP+ and t h e r e o x i d a t i o n by O 2 o f

t h i s compound. S ince hexok inase a l s o phosphory la tes

f r u c t o s e , t h i s s u b s t r a t e competes w i t h g lucose, r e s u l -

t i n g i n a decreased G 6 P f o r m a t i o n and thus i n a decre-

ased oxygen consumpt ion w i t h i n t h e membrane. There fo re

t h e sensor responds t o f r u c t o s e . F i g . 2 shows a t y p i c a l

response c u r v e t o g lucose and f r u c t o s e . I n t h e l i n e a r

r e g i o n o f t h e c o n c e n t r a t i o n dependences f o r t h e two sub-

s t r a t e s t h e r a t i o o f t h e response per c o n c e n t r a t i o n u n i t

was 11:l f o r g lucose t o f r u c t o s e .

The s e q u e n t i a l r e a c t i o n s i n t h e sensor were s t u d i e d

somewhat f u r t h e r . The dependence o f t h e response t o

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2160 SCHUBERT ET A L .

FIG. 2 . Response t o g l u c o s e and f r u c t o s e o f t h e bienzyme s e n s o r . The measuring b u f f e r contained 0 . 3 mM NADP’ and 3 mM A T P .

G 6 P , i . e . o f t h e G6P-OH r e a c t i o n , on NAUP’ c o n c e n t r a t i o n

i s shown i n F i g . 3. A p l a t e a u i s r e a c h e d a t 3 ,uM NADP’,

w h i c h i s i n t h e r a n g e o f t h e KM (NADP’) o f G6P-DH6. T h a t

t h i s low c o n c e n t r a t i o n i s s u f f i c i e n t f o r s a t u r a t i o n

seems t o be due t o t h e e f f e c t i v e c o f a c t o r r e c y c l i n g be-

tween NMP’ and t h e d e h y d r ~ g e n a s e ” ~ . A 1 1 f u r t h e r measure-

ments were c a r r i e d o u t w i t h 0.3 mM NADP’. Under t h e s e

c o n d i t i o n s t h e c a l i b r a t i o n c u r v e f o r G6P i s l i n e a r up t o

1.2 mM f i n a l c o n c e n t r a t i o n ( F i g . 4 ) . T h i s v a l u e by f a r

exceeds t h e Kh,(G6P) o f GGP-OH o f 20 ,uM, i n d i c a t i n g t h a t

t h e r e s p o n s e t o G6P i s d i f f u s i o n - c o n t r o l l e d .

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ENZYME ELECTRODE

n 1001 a

ELECTRODE

n 100 Q C Y

I I

2 4 6

2161

FIG. 3. Dependence of $he response to G6P of the bieneyme sensor on NADP concentration.

F o r measurement o f g l u c o s e and f r u c t o s e , r e s p e c t i v e l y ,

ATP must be p r e s e n t as hexok inase c o s u b s t r a t e . The de-

pendence o f t h e s i g n a l o b t a i n e d on a d d i t i o n o f 1 rnbl and

2 mM g l u c o s e on ATP c o n c e n t r a t i o n i s g i v e n i n F i g . 5 . A

t y p i c a l response c u r v e i s a l s o shown ( F i g . 5 B ) . The c u r -

0.4 0.8 1.2 1.6 2.0 glucose -6-phosphate CmM3

FIG. 4. Calibration curve f o r G6P of the bienzyme electrode.

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A:

B :

1 2 3 4 6 ATP CmMl

FIG. 5.

ATP g

B lrnC

1OnAI

H Smin

:oQ 1

f

Relationship between the minimum current (x) and the steady state current ( 0 , o ) , respectively, and the concentration of ATP in the measuring solution. Glucose was added to give 1 mM (-1 and 2 mM (---> final concentration.

Response to glucose of the bienzyme electrode in the presence of ATP.

r e n t decreased f o r 3 min and then inc reased t o r e a c h a

s teady s t a t e a f t e r 6 min. Wi th i n c r e a s i n g A T P concen-

t r a t i o n t h e d i f f e r e n c e between t h e m i n i m u m c u r r e n t and

t h e s teady s t a t e c u r r e n t decreased t o f i n a l l y d isappear

a t 3 mM A T P . I f a h i g h e r g lucose c o n c e n t r a t i o n , 2 mM, was

used, t h e s p i k e shape d isappeared a t h i g h e r A T P concen t ra -

t i o n . The s p i k e shape f o r m a t i o n migh t be caused by t h e

s low permeat ion o f A T P i n t o t h e enzme membrane. Obvious-

l y a f t e r g lucose a d d i t i o n t h e c o f a c t o r p r e s e n t i n t h e

enzyme l a y e r causes a f a s t oxygen consumption. D i f f u s i o n o f

A T P f rom t h e b u l k i s t o o s low t o m a i n t a i n t h i s h i g h r e -

a c t i o n r a t e as i s r e f l e c t e d by t h e decreased oxygen con-

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glucose Cml

FIG. 6. Dependence of the sensitivity to fructose of the bienzyme electrode on glucose concentration. 2 mM fructose was added to the measuring solution con- taining 3 mM ATP.

sumpt ion i n t h e s teady s t a t e . By i n c r e a s i n g t h e ATP b u l k

c o n c e n t r a t i o n i t s d i f f u s i o n i s a c c e l e r a t e d r e s u l t i n g i n

an ATP-independent s i g n a l h e i g h t . Fur te rmore , i f t h e se-

quence o f s u b s t r a t e a d d i t i o n was reve rsed , i . e . i f ATP

was added t o t h e background s o l u t i o n c o n t a i n i n g g lucose,

no peak was formed.

I n t h e presence o f 3 mM ATP t h e sensor s i g n a l depended

l i n e a r l y on g lucose c o n c e n t r a t i o n up t o 0.4 mM ( n o t shown).

The Kh, (g lucose ) o f hexokinase6 i s 0.1 mM. T h e r e f o r e d i f -

f u s i o n appears t o p l a y a s i g n i f i c a n t r o l e i n t h e g lucose

response, i . e . t h e sensor i s n o t k i n e t i c a l l y c o n t r o l l e d .

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2164

100

80

S 60- CI

U

.- 3 5 40-

rn 20-

.- u) C a

SCHUBERT ET AL.

-

-

I t I I I I

7.0 7.5 8.0 8.5 9.0 PH

FIG. 7. pH dependence of the fructose sensor signal.

The s e n s i t i v i t y t o f r u c t o s e o f t h e e l e c t r o d e was o p t i m a l

a t a g lucose c o n c e n t r a t i o n between 0.8 and 1.0 mM ( F i g . 6) .

There fo re f o r f r u c t o s e d e t e r m i n a t i o n s 3 mh4 ATP and 0.8 mM

g lucose were used. Keeping t h e r a t i o between them con-

s t a n t , a s h i f t o f t h e two compounds' c o n c e n t r a t i o n s d i d

n o t b r i n g about any s i g n i f i c a n t s e n s i t i v i t y enhancement.

The pH dependence o f t h e sensor f o r f r u c t o s e (F ig . 7)

i n d i c a t e s an o p t i m a l v a l u e a t pH 7.3 which i s c l o s e t o

t h e pH optimum o f hexokinase6. Under t h e recommended con-

d i t i o n s t h e c a l i b r a t i o n graph f o r f r u c t o s e was l i n e a r up t o

3 mM w i t h a d e t e c t i o n l i m i t o f 0.3 mM and a s l o p e between

17 and 21 ,uA mM'l ( F i g . 8). The u s e f u l l i f e t i m e o f t h e

f r u c t o s e sensor was one week.

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1 2 3 4 fructose ~ m M 3

FIG. 8. Calibration curve of the bienzyme electrode for fructose.

CONCLUSIONS

I n t h e p r e s e n t bienzyme e l e c t r o d e t h e s p e c i f i c a c t i v i t y

o f immob i l i zed GGP-DH i s s u f f i c i e n t l y h i g h t o o b t a i n d i f -

f u s i o n c o n t r o l . A l s o t h e g lucose response seems t o be

governed by s u b s t r a t e d i f f u s i o n . T h i s would i m p l y t h a t

an excess of hexok inase i s p r e s e n t i n t h e b i o c a t a l y t i c

membrane. I n s p i t e o f t h i s enzyme excess t h e sensor

r e a d i l y responds t o t h e a l t e r n a t i v e s u b s t r a t e , f r u c -

t ose , t h a t a c t s l i k e a c o m p e t i t i v e i n h i b i t o r on g l u -

cose p h o s p h o r y l a t i o n .

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The proposed sensor i s n o t on

f r u c t o s e d e t e r m i n a t i o n b u t may a

ATP measurement. Fur thermore, the

y a p p l i c a b l e t o

so be used f o r G6P and

sensor s i g n a l depends

on NADP' and g lucose. The use fu lness o f t h e sensor f o r

f r u c t o s e i s t h e r e f o r e r e s t r i c t e d by many p o t e n t i a l i n -

t e r f e r e n c e s . However, t h e i n t e r f e r e n c e s by ATP and NADP'

a r e avo ided by a d d i t i o n o f s a t u r a t i n g amounts o f these

compounds. A p a r t i c u l a r p rob lem i s posed by g lucose as

a major c o n s t i t u e n t o f many r e a l samples. T h i s compound

m igh t be removed u s i n g an enzymat ic a n t i - i n t e r f e r e n c e

membrane9. The d e f i n e d amount o f g lucose r e q u i r e d as

s u b s t r a t e i n f r u c t o s e measurement m igh t t hen be p r o -

duced w i t h i n t h e hexokinase-GGP-DH membrane by co im-

m o b i l i z e d glucoamylase''. I n t h i s case mal tose has t o

be added t o t h e measur ing b u f f e r . A t p r e s e n t t h i s m u l t i -

enzyme e l e c t r o d e i s under s tudy .

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9. F. S c h e l l e r % and R . Renneberg, A n a l . Chim. A c t a - 152

10. R . Renneberg, G. K a i s e r , F . S c h e l l e r and

Received September 10, 1986 Accepted September 18, 1986

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enzyme electrode for fructose, glucose-6-phosphate and atp determination

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