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Membrane potential of Bombyx mori silk fibroin membrane induced by an immobilized enzyme reaction
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Bioelectrochemistry and Bioenergetics, 26 (1991) 1 6 7 - 1 7 5 A section of J. Electroanal. Chem., a n d c o n s t i t u t i n g Vcl. 321 (199.1.) Elsevier Sequoia S.A.. L a u s a n n e
J E C BB 01383
M e m b r a n e potential of Bombyx mori silk fibroin m e m b r a n e i n d u c e d by an immobiliT.ed e n z y m e reaction Makoto Demura, Takashi Komura and Tetsuo Asakura
*
Faculty of Technology, Tokyo University of Agriculture end Technology, Nakamachi 2-Cl:ome, Koga,nei, Tokyo 184 (Japan) (Received 25 M a r c h 1991)
Abstract T h e m e m b r a n e p o t e n t i a l o f Bombyx mori silk fibroin m e m b r a n e i n d u c e d b y the r e a c t i o n o f glucose o x i d a s e ( G O ) which w a s i m m o b i l i z e d in the silk fibroin m e m b r a n e , h a s b e e n m e a s u r e d . T h e m e m b r a n e p o t e n t i a l increased r a p i d l y a f t e r a d d i t i o n o f the substrate, glucose, a n d r e a c h e d a p l a t e a u . T h e d e g r e e o f r e s p o n s e d e p e n d e d o n the c o n c e n t r a t i o n o f glucose. F r o m direct o b s e r v a t i o n o f the 13C N M R s p e c t r a o f the p r o d u c t o f the e n z y m e reaction in the silk fibroin m e m b r a n e , it was c o n c l u d e d t h a t the. g l u c o n o l a c t o n e generated f r o m the glucose w a s c o m p l e t e l y c o n v e r t e d to gluconic acid in the silk fibroin m e m b r a n e . I n addition, a greater p H difference b e t w e e n the m e m b r a n e surfaces a f t e r the e n z y m e r e a c t i o n w a s d e t e r m i n e d . I t was f o u n d t h a t the effective anionic fixed c h a r g e d e n s i t y o f silk fibroin m e m b r a n e d e c r e a s e d with increasing gluconic acid c o n c e n t r a t i o n in the m e m b r a n e .
INTRODUCTION
Silk fibroin p r e p a r e d f r o m Bombyx mori c o c o o n s has b e e n appfied successfully as a support for enzyme immobiliTation [1-12]. O n e of the merits o f silk f i b r o i n as a n enzyme i m m o b i l i z a t i o n s u p p o r t is that the enzymes c a n be mildly e n t r a p p e d i n t o the silk fibroin m e m b r a n e w i t h o u t a n y use o f chemical reagents. N a m e l y , the structural t r a n s i t i o n of the fibroin f r o m r a n d o m coil to anti-parallel ~-sheet occurs readily u p o n physical t r e a t m e n t only, such as stretching, compressing, s t a n d i n g u n d e r high h u m i d i t y a n d i m m e r s i n g in a n alcohol aqueous solution, resulting in t h e simultaneous insolubi!iTation o f the m e m b r a n e a n d the i m m o b i l i z a t i o n o f e n z y m e [13,14]. In addition, a sensor assembled f r o m a n o x y g e n electrode using glucose oxidase (GO) i m m o b i l i z e d w i t h i n a s~lk fibroin m e m b r a n e has been d e v e l o p e d
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© 1991 - Elsevier S e q u o i a S.A. All rights reserved
i6s T h e m e m b r a n e p o t e n t i a l i n d u c e d b y the r e a c t i o n in a n d / o r o n a m e m b r a n e h a s b e e n m e a s u r e d w i t h s o m e e n z y m e i m m o b i l i z e d m e m b r a n e s [15-18]. H o w e v e r , these n e e d large a m o u n t s o f e n z y m e a n d the s t r u c t u r a l effect o f the e n z y m e s u p p o r t s o n t h e m e m b r a n e p o t e n t i a l i n d u c e d b y the i m m o b i l i z e d e n z y m e r e a c t i o n w a s n o t discussed. I n this paper, we will r e p o r t the m e m b r a n e p o t e n t i a l o f B. mori silk f i b r o i n m e m b r a n e i n d u c e d b y G O i m m o b i l i z e d in t h e m e m b r a n e . T h e d e g r e e o f dissociat i o n o f the ionic p r o d u c t of t h e ( 3 0 r e a c t i o n in the m e m b r a n e p h a s e is d e t e r m i n e d b y 13C n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) s p e c t r o s c o p y . T h e b a s i c m e c h a n i s m o f t h e g e n e r a t i o n of the m e m b r a n e p o t e n t i a l a f t e r the e n z y m e r e a c t i o n will b e d e d u c e d f r o m the b e h a v i o r of t h e p r o d u c t s , t h a t is, their i n f l u e n c e o n b o t h tile e n z y m e r e a c t i o n a n d the s t r u c t u r e of the silk f i b r o i n m e m b r a n e as a s u p p o r t . T h e g e n e r a t i o n o f t h e m e m b r a n e p o t e n t i a l o f t h e silk f i b r o i n m e m b r a n e as r e g a r d s t h e c o n c e n t r a t i o n p o t e n t i a l w i t h a l k a l i n e ions h a s b e e n i n v e s t i g a t e d p r e v i o u s l y [19,20]. i t is f o u n d t h a t g e n e r a t i o n of t h e m e m b r a n e p o t e n t i a l b y t h e r e a c t i o n of G O i m m o b i l i z e d in t h e silk fibroin m e m b r a n e is d u e to t h e p r o d u c t i o n of i o n i c species b y the i m m o b i l i z e d enzyme. EXPERIMENTAL
Preparation of the GO immobilized silk fibroin membrane T h e silk f i b r o i n a q u e o u s s o l u t i o n w a s p r e p a r e d f r o m Bombyx mori c o c o o n s as d e s c r i b e d p r e v i o u s l y [9,21]. 3 w / v % of t h e silk f i b r o i n s o l u t i o n w a s m i x e d m i l d l y w i t h a given a m o u n t o f G O (derived f r o m Aspergiilus niger, W A K O P u r e R e a g e n t s Co.) a q u e o u s solution. T h i s m i x t u r e w a s c a s t o n a n a c r y l i c p l a s t i c p l a t e , a n d d r i e d at 2 0 ° C u n d e r 50% relative h u m i d i t y for 48 h. T h e d r i e d m e m b r a n e w a s i n s o l u b i l i z e d b y i m m e r s i o n in 80% m e t h a n o l a q u e o u s s o l u t i o n for 1 h, a n d t h e n w a s h e d w i t h distilled w a t e r [11]. T h e G O i m m o b i l i z e d silk f i b r o i n m e m b r a n e w a s s t o r e d i n a d r y s t a t e at 4 ° C u n t i l m e a s u r e m e n t s . T h e t h i c k n e s s o f t h e m e m b r a n e w a s ca. 5 0 / ~ m .
Measurement of membrane potential T h e G O i m m o b i l i z e d silk f i b r o i n m e m b r a n e w a s s u f f i c i e n t l y s w o l l e n in d i s t i l l e d w a t e r a n d t h e n the m e m b r a n e w a s set p o s i t i o n e d b e t w e e n the cells w i t h silicone sheets as s h o w n in Fig. 1. T h e v o l u m e of e a c h c o m p a r t m e n t w a s 80 m l a n d t h e effective r e a c t i o n a r e a o f t h e m e m b r a n e w a s 3.14 c m 2. A n e l e c t r o m e t e r ( M o d e l T R 8652, A d v a n t e s t Co., J a p a n ) w a s c o n n e c t e d to each c o m p a r t m e n t w i t h A g / A g C l electrodes t h r o u g h salt bridges, A f t e r t h e cell was filled w i t h 0.1 m M p o t a s s i u m p h o s p h a t e b u f f e r s c ! u t i o n ( p H 7), a given a m o u n t of g l u c o s e a q u e o u s s o l u t i o n (1 M ) w a s a d d e d to the left c o m p a r t m e n t a n d the p o t e n t i a l d i f f e r e n c e b e t w e e n t h e t w o c o m p a r t m e n t s w a s m e a s u r e d at 2 5 ° C .
13C N M R measurement ~ a C N M R s p e c t r a w e r e r e c o r d e d at 2 5 ° C w i t h a J E O L F X - 9 0 Q N M R s p e c t r o m e ter o p e r a t i n g at 22.49 M H z . T h e p r o d u c t of t h e ( 3 0 r e a c t i o n , g l u c o n o l a e t o n e , w a s
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i o I-::-0---! o-I Fig. 1. S c h e m e o f the apparatus for m e a s u r e m e n t o f the m e m b r a n e potential with the G O reaction in the silk fibroin membrane. (1) Right c o m p a r t m e n t , (2} left c o m p a r t m e n t , {3) salt bridge, (4) substrate solution, (5) G O i m m o b i l i z e d silk fibroin m e m b r a n e , (6} Stirrer.
measured in the aqueous solution, where the p H was adjusted with 0.1 M s o d i u m h y d r o x i d e a n d h y d r o c h l o r i c acid aqueous solutions. Moreover, the t3C N M R spectrum of the p r o d u c t in the silk fibroin m e m b r a n e after the G O reaction was observed directly. After the ( 3 0 immobilized m e m b r a n e was immersed in 0.5 M glucose aqueous solution ( p H 7) for 30 rain, the m e m b r a n e was picked up, a n d then measured. Measurement o f p H after the GO reaction T h e p H c h a n g e of the G O immobilized silk fibroin m e m b r a n e after the e n z y m e reaction was measured b y the following m e t h o d . T h e G O immobilized silk fibroin m e m b r a n e was d i p p e d in 0.1 m M p o t a s s i u m p h o s p h a t e buffer ( p H 7) overnight. T h e n the m e m b r a n e was a t t a c h e d to a glass p H electrode ( M o d e l TP-101, T o k o Chemical L a b o r a t o r i e s Co., T o k y o , J a p a n ) with a n y l o n net a n d an O-ring. A given a m o u n t of glucose solution (1 M) was injected into the reaction c h a m b e r with the assembled p H electrode. T h e n the p H c h a n g e was recorded. T h e change of p H in the silk fibroin m e m b r a n e was also m e a s u r e d with a p H indicator, 3-sulfo-2,6-dichloro-3",3"-dimethyl-4"-hydroxyfuchsone-5",5"-dicarboxylic acid, trisodium salt ( C h r o m a z u r o l S, D o j i n d o Laboratories, ~ , u m a m o t o , Japan), by m o n i t o r i n g the a b s o r b a n c e at 430 n m b y m e a n s of a n U V s p e c t r o m e t e r (U-3200, Hitachi) equipped with a t h e r m o s t a t e d cell holder. Determination of the f i x e d charge density o f the silk fibroin membrane . . . . . . T h e m e m b r a n e p o t e n t i a l was measured at a given p H b y c h a n g i n g the concentration of KC! in each c o m p a r t m e n t while m a i n t a i n i n g the c o n c e n t r a t i o n ratio of KCI
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Fig. 2. Typical response of the membrane potential with the reaction of 2% GO immobilized in the silk fibroin membrane after injection of glucose (final concentration = 1 raM). at 2:1. The fixed charge density was calculated p o t e n t i a l a s a f u n c t i o n o f p H [19].
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RESULTS AND DISCUSSION
M e m b r a n e potential induced by the G O reaction The membrane potential of the GO immobilized silk fibroin membrane during the enzyme reaction was measured. Figure 2 depicts a typical response of the membrane potential of the GO immobilized silk fibroin membrane after injection of t h e s u b s t r a t e . I n t h i s c a s e , t h e c o n c e n t r a t i o n o f p o t a s s i u m p h o s p h a t e in e a c h cell w a s t h e s a m e a n d d i d n o t c h a n g e d u r i n g t h e i n j e c t i o n . It h a s b e e n r e p o r t e d p r e v i o u s l y t h a t n o l e a k a g e o f G O f r o m t h e silk f i b r o i n m e m b r a n e w a s o b s e r v e d [5]. T h e m e m b r a n e p o t e n t i a l i n c r e a s e d g r a d u a l l y a n d r e a c h e d a p l a t e a u , as s h o w n in P i g . 2. I n t h e a b s e n c e o f G O , n o m e m b r a n e p o t e n t i a l w a s o b s e r v e d a f t e r t h e injection of glucose solution. Figure 3 shows a plot of A E, the membrane potential in t h e s t e a d y s t a t e , a g a i n s t t h e g l u c o s e c o n c e n t r a t i o n o f t h e s o l u t i o n in t h e l e f t compartment. The potential increased with increasing glucose concentration in the range of 0 to 5 mM. Previously, the membrane potential of an enzyme immobilized m e m b r a n e h a d b e e n m e a s u r e d w i t h a n a l b u m i n m e m b r a n e b e a t i n g u r e a s e [22] a n d
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Fig. 4. laC NMR spectra of products of the GO reaction. (31uconolactone aqueous solution at two pHs: (A) 6.8 and (B) 3.0. (C) Porous silk fibroin membrane after immobilized GO reaction. C1-C6: (31uconic acid, (A) gluconolactone. w i t h a c e t y l c h o l i n e s t e r a s e i m m o b i l i z e d ossein g e l a t i n m e m b r a n e o r a l b u m i n m e m b r a n e [181. T h e f o r m e r w a s m e a s u r e d w i t h d i f f e r e n t p h o s p h a t e b u f f e r c o n c e n t r a tions o n e a c h side o f t h e m e m b r a n e . T h e m e m b r a n e p o t e n t i a l c h a n g e s w e r e b a s e d o n t he r e c i p r o c a l e f f e c t b e t w e e n e n z y m e a c t i v i t y a n d t h e a p p e a r a n c e o f i o n s a f t e r t h e e n z y m e r e a c t i o n , i.e., a m o d i f i c a t i o n o f t h e local p H a n d a f e e d b a c k a c t i o n o f b u f f e r d i f f u s i o n f r o m t he o u t s i d e s o l u t i o n . T h e l a t t e r s t u d y also p o i n t e d o u t t h e e f f e c t o n t h e m e m b r a n e p o t e n t i a l o f i o n i c sp ecies p r o d u c e d b y t h e e n z y m e . H o w ever, it is i n s u f f i c i e n t to u n d e r s t a n d th e d e t a i l e d info~',~ation c o n c e r n i n g t h e effect of products on the enzyme support.
SaC N M R observation o f the GO reaction in the silk fibroin membrane It is n e c e s s a r y t o p a y a t t e n t i o n to th e i o n i c p r o d u c t , g l u c o n i c acid, b e c a u s e t h e i o n i c p r o d u c t in t h e m e m b r a n e m a y g e n e r a t e th e i o n d i f f u s i o n in th e m e m b r a n e a n d / o r t h e local p H c h a n g e in t h e m e m b r a n e . I n a d d i t i o n , t h e fine s t r u c t u r e o f t h e silk f i b r o i n m e m b r a n e is c o n s i d e r e d to b e a f f e c t e d b y t h e i o n i c p r o d u c t . H o w e v e r , th e s t a t e of t h e i o n i c species p r o d u c e d in t h e m e m b r a n e w a s n o t o b s e r v e d d i r e c t l y in spi t e o f t h e i m p o r t a n c e o f t h e i o n i c p r o d u c t . T h e r e f o r e , w e o b s e r v e d t h e s t a t e o f t h e p r o d u c t s a f t e r t h e e n z y m e r e a c t i o n in t h e silk f i b r o i n m e m b r a n e u s i n g ~aC N M R . F i g u r e s 4 A a n d B s h o w ~aC N M R s p e c t r a o f g l u c o n o l a c t o n e a q u e o u s s o l u t i o n at t w o p H s , 6.8 a n d 3.0, respectively. All p e a k i n t e n s i t i e s a t t r i b u t a b l e to g l u c o n i c a c i d a r e
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Fig. 5. C o n c e n t r a t i o n vs. m e m b r a n e potential o f the silk fibroin m e m b r a n e with gluconic acid p r o d u c e d
from gluconolactone, and of KCI. The concentration ratio (c2/c~) was changed from 1 to 2. (e) Gluconic acid (cl = 0.01 M), (o) gluconic acid (ca ----0.001 M). (zx) KCI (c i ----0.01 M). s t r o n g e r t h a n t h o s e of g l u c o n o l a c t o n e c a r b o n s e v e n at p H = 3.0. F u r t h e r , Fig. 4 C d e p i c t s the 13C N M R s p e c t r u m o f the p r o d u c t in p o r o u s G O i m m o b i l i z e d m e m b r a n e g e n e r a t e d by the i m m o b i l i z e d e n z y m e in t h e m e m b r a n e . T h e p e a k s can b e a s s i g n e d exclusively to the c a r b o n s o f g l u c o n i c acid, a l t h o u g h t h e p e a k s are b r o a d b e c a u s e o f the low m o b i l i t y o f t h e p r o d u c t s r a t h e r t h a n t h e high m o b i l i t y o f t h o s e in s o l u t i o n . U n d e r t h e s e c o n d i t i o n s , t h e c a r b o n s o f silk fibroin w e r e n o t o b s e r v e d . T h a s , it is c o n c l u d e d that g l u c o n o l a c t o n e , w h i c h is the p r o d u c t o f t h e G O r e a c t i o n , is r a p i d l y h y d r o l y z e d to g l u c o n i c acid in t h e silk fibroin m e m b r a n e . In a d d i t i o n , p H t i t r a t i o n o f g l u c o n i e acid s u g g e s t e d t h a t t h e p H in the G O i m m o b i l i z e d m e m b r a n e is s h i f t e d to the acidic side b y t h e e n z y m e reaction.
Effects of gluconic acid on the membrane potential of the silk fibroin membrane F i g u r e 5 d e p i c t s the c o n c e n t r a t i o n m e m b r a n e p o t e n t i a l o f the silk f i b r o i n m e m b r a n e w i t h g l u c o n i c acid p r o d u c e d f r o m g l u c o n o l a c t o n e . This i n d i c a t e s t h a t t h e a d d i t i o n o f g l u c o n i c acid to o n e side o f t h e silk f i b r o i n m e m b r a n e g e n e r a t e s a m e m b r a n e p o t e n t i a l a n d t h a t t h e p o l a r i t y is t h e s a m e as with KCl. H o w e v e r , t h e m e m b r a n e p o t e n t i a l d i f f e r e n c e s u s i n g g l u c o n i e acid are small c o m p a r e d w i t h t h o s e u s i n g KCI, s u g g e s t i n g an effect o f p H c h a n g e o n t h e m e m b r a n e p o t e n t i a l . W h e n t h e g l u c o n i c acid is p r e f e r e n t i a l l y p r o d u c e d w i t h t h e i m m o b i l i z e d G O r e a c t i o n at o n e side o f t h e m e m b r a n e , this i o n i c species m a y d i f f u s e across t h e silk f i b r o i n m e m b r a n e . It is e x p e c t e d that a p H c h a n g e o c c u r s locally, c a u s e d b y t h e p r e s e n c e o f g l u c o n i c acid. Such a p H c h a n g e o n t h e m e m b r a n e s u r f a c e a n d in t h e
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8
Fig. 6. Plots of the rate of pH change of the GO immobilized silk fibroin membrane against the concentration of glucose. (o) 0.2% GO, (A) 1.0% GO, (~) 2.0% GO.
silk f i b r o i n m e m b r a n e was m e a s u r e d with b o t h a p H glass e l e c t r o d e a n d U V m e t h o d s . T h e f o r m e r m e t h o d gives p H i n f o r m a t i o n c o n c e r n i n g t h e s u r f a c e n o t in c o n t a c t w i t h t h e s u b s t r a t e directly, w h i l e the l a t t e r o n e gives t h e i n f o r m a t i o n o f p H c h a n g e o n t h e o t h e r m e m b r a n e s u r f a c e a n d in t h e m e m b r a n e . F i g u r e 6 s h o w s the r e l a t i o n s h i p b e t w e e n t h e rate o f p H c h a n g e in the s t e a d y s t a t e a n d t h e g l u c o s e c o n c e n t r a t i o n in t h e r e a c t i o n c h a m b e r . D u r i n g t h e G O r e a c t i o n , t h e p H o n the m e m b r a n e surface n o t in c o n t a c t w i t h t h e s u b s t r a t e was s h i f t e d to t h e a c i d i c side a n d t h e rate of t h e p H c h a n g e d e p e n d e d o n t h e c o n c e n t r a t i o n o f g l u c o s e in t h e r e a c t i o n c h a m b e r . T h e p l o t s are r e g a r d e d as s t r a i g h t lines, t h e slopes o f w h i c h i n c r e a s e w i t h i n c r e a s i n g c o n t e n t o f i m m o b i l i z e d ( 3 0 . T h e s l o p e s are 0.33 x 10 - a , 4.17 x 10 - 3 a n d 12.3 x 10 - 3 A p H m i n -1 m M - t , for G O c o n t e n t s o f 0.2%, 1.0% a n d 2.0%, respectively, w h i c h c o r r e s p o n d to t h e a m o u n t o f g l u c o n i c acid p r o d u c e d b y t h e ( 3 0 r e a c t i o n in t h e silk f i b r o i n m e m b r a n e . T h e p H c h a n g e o n t h e m e m b r a n e s u r f a c e in c o n t a c t w i t h t h e s u b s t r a t e w a s also e s t i m a t e d , f r o m t h e U V m e t h o d . T h e a b s o r b a n c e o f a p H i n d i c a t o r , C h r o m a z u r o i S, in t h e p r e s e n c e o f b o t h a G O i m m o b i l i z e d m e m b r a n e a n d g l u c o s e was r e c o r d e d c o n t i n u o u s l y a n d t h e n t h e initial rate o f t h e p H c h a n g e w i t h t h e e n z y m e r e a c t i o n w a s d e t e r m i n e d b y u s i n g t h e c a l i b r a t i o n curve. F o r a G O c o n t e n t o f 0.2%, t h e rate w a s 9.8 x 10 - 3 A p H m i n -~ m M -1. T h u s , t h e p H c h a n g e at t h e surface in c o n t a c t w i t h t h e s u b s t a n c e w a s m u c h larger t h a n t h a t o b s e r v e d u s i n g t h e p H e l e c t r o d e . T h i s result i n d i c a t e s that the p r o d u c t i o n o f g l u c o n i c acid b y t h e i m m o b i l i z e d ( 3 0 r e a c t i o n m a y c h a n g e the p H o f b o t h t h e i n n e r a n d s u r f a c e r e g i o n s o f t h e m e m b r a n e a n d t h a t t h e c o n c e n t r a t i o n of p r o d u c t at b o t h surfaces o f t h e m e m b r a n e is n o t equal. T h e s e results agree w i t h the c o n c e n t r a t i o n m e m b r a n e p o t e n t i a l w i t h g l u c o n i c acid, t h e ~3C N M R o b s e r v a t i o n o f g l u c o n i c acid a n d also w i t h a t h e o r e t i c a l s t u d y o f t h e g e n e r a l t r a n s p o r t of t h e p r o d u c t s o f an e n z y m e m e m b r a n e [23]. T h e n o n - u n i f o r m d i s t r i b u t i o n o f t h e i o n i c species o v e r t h e t w o m e m b r a n e surfaces is c o n s i d e r e d t 0 r e s u l t n o t o n l y from i o n d i f f u s i o n b u t also f r o m t h e a s y m m e t r i c c h a n g e o f t h e m i c r o e n v i r o n m e n t of the silk f i b r o i n m e m b r a n e (see b e l o w ) .
174
20
0
i IL.,IPJ~=
4.
|
5
6
t
i
7
8
Fig. 7. Fixed charge density of various silk fibroin membranes as a function of pH. McIlvaine buffer concentration was 0.01 M. (o) GO immobilized silk fibroin membrane, (o) silk fibroin membrane.
T h e effective fixed c h a r g e d e n s i t y o f t h e silk fib ro in is e x p e c t e d to b e a f f e c t e d by t h e g l u c o n i c a c i d d u r i n g t h e e n z y m e r e a c t i o n in th e m e m b r a n e . F i g u r e 7 s h o w s p l o t s o f t h e fixed c h a r g e d e n s i t y as a f u n c t i o n of p H . T h e silk fib ro in m e m b r a n e h a s n e t positive c h a r g e s in t h e a c i d i c r e g i o n a n d n e g a t i v e o n e s in t h e b a s i c r e g i o n relative to t h e isoelectric p o i n t , w h i c h is p H 4.3 for th e o r i g i n a l silk fib ro in m e m b r a n e a n d p H 4.5 for the ( 3 0 i m m o b i l i z e d silk f i b r o i n m e m b r a n e . I n p a r t i c u l a r , t h e e f f e c t i v e fixed c h a r g e d e n s i t y c h a n g e d r e m a r k a b l y in t h e p H r a n g e f r o m 7 to t h e p H o f t h e isoe l e c t ri c p o i n t . T h e r e f o r e , this c h a n g e relates to t h e g e n e r a t i o n o f the D o n n a n p o t e n t i a l o n e a c h s u r f a c e of t h e ( 3 0 i m m o b i l i z e d silk f i b r o i n m e m b r a n e . In a d d i t i o n , t he fine s t r u c t u r e o f t he silk f i b r o i n m e m b r a n e is e x p e c t e d to b e i n f l u e n c e d b y t h e i n t e r a c t i o n b e t w e e n g l u c o n i c a c i d a n d t h e silk f i b r o i n e n z y m e s u p p o r t , s u g g e s t i n g a n effect o f m e m b r a n e s t r u c t u r e o n t h e m e m b r a n e p o t e n t i a l c o u p l e d to the immobilized enzyme reaction. I n g e n e r a l , t h e m e m b r a n e p o t e n t i a l d i f f e r e n c e w i t h a n artificial fixed c h a r g e m e m b r a n e s u c h as a silk f i b r o i n m e m b r a n e is e x p r e s s e d as fo llo w s [24]: A E AEDon.~ . + A E d i f f u s i o n , w h e r e A E D o . ~ ~ a n d AEdiffu~io n a r e th e D o n n a n a n d d i f f u s i o n p o t e n t i a l s , respectively. T h e f o r m e r p o t e n t i a l is t h e s u m o f t h e t w o s u r f a c e p o t e n tials at b o t h sides o f t he m e m b r a n e a n d t h e l a t t e r is t h e d i f f u s i o n p o t e n t i a l in t h e m e m b r a n e . T h e e n z y m e r e a c t i o n in t h e m e m b r a n e m a y c a u s e a c h a n g e in b o t h AEDo~na.~ a n d AEd{ff,,~io.. It is p a r t i c u l a r l y i m p o r t a n t to reveal t h e b e h a v i o r o f i o n i c spe c i e s a f t e r the e n z y m e r e a c t i o n in t h e silk fib ro in m e m b r a n e . It will t h e r e f o r e b e n e c e s s a r y t o i n v e s t i g a t e t h e m o b i l i t y o f ionic species in t h e silk f i b r o i n m e m b r a n e a n d the i n t e r a c t i o n b e t w e e n the p r o d u c t a n d th e silk fibro.ln in o r d e r to i m p r o v e t h e r e s p o n s e a n d to d e s i g n a n effective s t r u c t u r e for th e silk f i b r o i n m e m b r a n e as a n enzyme support. •. . . . .
175 REFERENCES 1 L. Grasset, D. Cordier and A. Ville, Biotechnol. Bioeng., 19 (1977) 611. 2 L. Grasset, D. Cordier and A. Ville, Process Biochem., 14 (1979) 2. 3 S. Miyairi, M. Sugiura and F. Fukui, Agric. Biol. Chem., 42 (1978) 1661. 4 D. Cordier, R. Couturier, L. Grasset and A. Viile, Enzyme Microb. Technoi.o ,5 (1982) 249. 5 A. Kuzuhara, T. Asakura, R. T o m o d a and T. Matsunaga, J.Biotechnoi., 5 (1987) 199. 6 T. Asakura, Bioindustry, 4 (1987) 36. 7 T. Asakura, H. Yoshimizu, A. Kuzuhara and T. Matsunaga, J. Seric. Sci. Jpn., 57 (1988) 203. 8 T. Asakura, J. Kanetake, and M. Demura, J. Polym. Plast. Tecknoi. Eng., 28 (1989) 453. 9 M. Demura and T. Asakura, Biotechnol. Biocng., 33 (1989) 598. 10 M. Demura, T. Asakura, E. Nakamura and H. Tamura, J. Biotechnol., 10 (1989) 113. 11 M, Demura, T. Asakura and T. Kuroo, Biosensors, ,5 (~989) 361. 12 H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (I990) 127. 13 H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 1745. 14 T. Asakura, H. Yoshimizu and M. Kakizaki, Biotechnol. Bioeng., 35 (1990) 511. 15 R. To r and A. Freeman, Anal. Chem., 58 (1979) 1042. 16 T. Shinbo, M. Sugiura and N. Kamo, Anal. Chem., 51 (1979) 100. 17 R.H. William and H.B. Halsall, Anal. Chem., 57 (1985) 1321A. 18 A. Friboulet and D. Thomas, Biophys. Chem., 16 (1982) 153. 19 M. Sugiura, Nippon Nogei Kagaku Kaishi, 47 (1973) 563. 20 M. Demura, A, Kitamura, A. Shibamoto and T. Asakura, J. Appl. Polym. Sci., 36 (1988) 535. 21 T. Asakura, Y. Watanabe, A. Uchida and H. Minagawa, Macromolecules, 17 (1984) 1075. 22 A. David, M. Metayer, D. Thomas and G. Broum, J. Membrane Biol., 18 (1974) 113. 23 T. Ciftci and W.R. Vieth, J. Mol. Catal., 8 (1980) 455. 24 Y. Kobatakeo K. K.urihara N. K am o in T. Takamura and A. Kozawa (Eds.), Surface Electrochemistry. Advances and Concepts, Japan Scientific Societies Press, Tokyo, 1978, p. 1.
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Bioelectrochemistry and Bioenergetics, 26 (1991) 1 6 7 - 1 7 5 A section of J. Electroanal. Chem., a n d c o n s t i t u t i n g Vcl. 321 (199.1.) Elsevier Sequoia S.A.. L a u s a n n e
J E C BB 01383
M e m b r a n e potential of Bombyx mori silk fibroin m e m b r a n e i n d u c e d by an immobiliT.ed e n z y m e reaction Makoto Demura, Takashi Komura and Tetsuo Asakura
*
Faculty of Technology, Tokyo University of Agriculture end Technology, Nakamachi 2-Cl:ome, Koga,nei, Tokyo 184 (Japan) (Received 25 M a r c h 1991)
Abstract T h e m e m b r a n e p o t e n t i a l o f Bombyx mori silk fibroin m e m b r a n e i n d u c e d b y the r e a c t i o n o f glucose o x i d a s e ( G O ) which w a s i m m o b i l i z e d in the silk fibroin m e m b r a n e , h a s b e e n m e a s u r e d . T h e m e m b r a n e p o t e n t i a l increased r a p i d l y a f t e r a d d i t i o n o f the substrate, glucose, a n d r e a c h e d a p l a t e a u . T h e d e g r e e o f r e s p o n s e d e p e n d e d o n the c o n c e n t r a t i o n o f glucose. F r o m direct o b s e r v a t i o n o f the 13C N M R s p e c t r a o f the p r o d u c t o f the e n z y m e reaction in the silk fibroin m e m b r a n e , it was c o n c l u d e d t h a t the. g l u c o n o l a c t o n e generated f r o m the glucose w a s c o m p l e t e l y c o n v e r t e d to gluconic acid in the silk fibroin m e m b r a n e . I n addition, a greater p H difference b e t w e e n the m e m b r a n e surfaces a f t e r the e n z y m e r e a c t i o n w a s d e t e r m i n e d . I t was f o u n d t h a t the effective anionic fixed c h a r g e d e n s i t y o f silk fibroin m e m b r a n e d e c r e a s e d with increasing gluconic acid c o n c e n t r a t i o n in the m e m b r a n e .
INTRODUCTION
Silk fibroin p r e p a r e d f r o m Bombyx mori c o c o o n s has b e e n appfied successfully as a support for enzyme immobiliTation [1-12]. O n e of the merits o f silk f i b r o i n as a n enzyme i m m o b i l i z a t i o n s u p p o r t is that the enzymes c a n be mildly e n t r a p p e d i n t o the silk fibroin m e m b r a n e w i t h o u t a n y use o f chemical reagents. N a m e l y , the structural t r a n s i t i o n of the fibroin f r o m r a n d o m coil to anti-parallel ~-sheet occurs readily u p o n physical t r e a t m e n t only, such as stretching, compressing, s t a n d i n g u n d e r high h u m i d i t y a n d i m m e r s i n g in a n alcohol aqueous solution, resulting in t h e simultaneous insolubi!iTation o f the m e m b r a n e a n d the i m m o b i l i z a t i o n o f e n z y m e [13,14]. In addition, a sensor assembled f r o m a n o x y g e n electrode using glucose oxidase (GO) i m m o b i l i z e d w i t h i n a s~lk fibroin m e m b r a n e has been d e v e l o p e d
[9,111. * T o w h o m c o r r e s p o n d e n c e should b e a d d r e s s e d . 0302-4598/91/$03.50
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© 1991 - Elsevier S e q u o i a S.A. All rights reserved
i6s T h e m e m b r a n e p o t e n t i a l i n d u c e d b y the r e a c t i o n in a n d / o r o n a m e m b r a n e h a s b e e n m e a s u r e d w i t h s o m e e n z y m e i m m o b i l i z e d m e m b r a n e s [15-18]. H o w e v e r , these n e e d large a m o u n t s o f e n z y m e a n d the s t r u c t u r a l effect o f the e n z y m e s u p p o r t s o n t h e m e m b r a n e p o t e n t i a l i n d u c e d b y the i m m o b i l i z e d e n z y m e r e a c t i o n w a s n o t discussed. I n this paper, we will r e p o r t the m e m b r a n e p o t e n t i a l o f B. mori silk f i b r o i n m e m b r a n e i n d u c e d b y G O i m m o b i l i z e d in t h e m e m b r a n e . T h e d e g r e e o f dissociat i o n o f the ionic p r o d u c t of t h e ( 3 0 r e a c t i o n in the m e m b r a n e p h a s e is d e t e r m i n e d b y 13C n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) s p e c t r o s c o p y . T h e b a s i c m e c h a n i s m o f t h e g e n e r a t i o n of the m e m b r a n e p o t e n t i a l a f t e r the e n z y m e r e a c t i o n will b e d e d u c e d f r o m the b e h a v i o r of t h e p r o d u c t s , t h a t is, their i n f l u e n c e o n b o t h tile e n z y m e r e a c t i o n a n d the s t r u c t u r e of the silk f i b r o i n m e m b r a n e as a s u p p o r t . T h e g e n e r a t i o n o f t h e m e m b r a n e p o t e n t i a l o f t h e silk f i b r o i n m e m b r a n e as r e g a r d s t h e c o n c e n t r a t i o n p o t e n t i a l w i t h a l k a l i n e ions h a s b e e n i n v e s t i g a t e d p r e v i o u s l y [19,20]. i t is f o u n d t h a t g e n e r a t i o n of t h e m e m b r a n e p o t e n t i a l b y t h e r e a c t i o n of G O i m m o b i l i z e d in t h e silk fibroin m e m b r a n e is d u e to t h e p r o d u c t i o n of i o n i c species b y the i m m o b i l i z e d enzyme. EXPERIMENTAL
Preparation of the GO immobilized silk fibroin membrane T h e silk f i b r o i n a q u e o u s s o l u t i o n w a s p r e p a r e d f r o m Bombyx mori c o c o o n s as d e s c r i b e d p r e v i o u s l y [9,21]. 3 w / v % of t h e silk f i b r o i n s o l u t i o n w a s m i x e d m i l d l y w i t h a given a m o u n t o f G O (derived f r o m Aspergiilus niger, W A K O P u r e R e a g e n t s Co.) a q u e o u s solution. T h i s m i x t u r e w a s c a s t o n a n a c r y l i c p l a s t i c p l a t e , a n d d r i e d at 2 0 ° C u n d e r 50% relative h u m i d i t y for 48 h. T h e d r i e d m e m b r a n e w a s i n s o l u b i l i z e d b y i m m e r s i o n in 80% m e t h a n o l a q u e o u s s o l u t i o n for 1 h, a n d t h e n w a s h e d w i t h distilled w a t e r [11]. T h e G O i m m o b i l i z e d silk f i b r o i n m e m b r a n e w a s s t o r e d i n a d r y s t a t e at 4 ° C u n t i l m e a s u r e m e n t s . T h e t h i c k n e s s o f t h e m e m b r a n e w a s ca. 5 0 / ~ m .
Measurement of membrane potential T h e G O i m m o b i l i z e d silk f i b r o i n m e m b r a n e w a s s u f f i c i e n t l y s w o l l e n in d i s t i l l e d w a t e r a n d t h e n the m e m b r a n e w a s set p o s i t i o n e d b e t w e e n the cells w i t h silicone sheets as s h o w n in Fig. 1. T h e v o l u m e of e a c h c o m p a r t m e n t w a s 80 m l a n d t h e effective r e a c t i o n a r e a o f t h e m e m b r a n e w a s 3.14 c m 2. A n e l e c t r o m e t e r ( M o d e l T R 8652, A d v a n t e s t Co., J a p a n ) w a s c o n n e c t e d to each c o m p a r t m e n t w i t h A g / A g C l electrodes t h r o u g h salt bridges, A f t e r t h e cell was filled w i t h 0.1 m M p o t a s s i u m p h o s p h a t e b u f f e r s c ! u t i o n ( p H 7), a given a m o u n t of g l u c o s e a q u e o u s s o l u t i o n (1 M ) w a s a d d e d to the left c o m p a r t m e n t a n d the p o t e n t i a l d i f f e r e n c e b e t w e e n t h e t w o c o m p a r t m e n t s w a s m e a s u r e d at 2 5 ° C .
13C N M R measurement ~ a C N M R s p e c t r a w e r e r e c o r d e d at 2 5 ° C w i t h a J E O L F X - 9 0 Q N M R s p e c t r o m e ter o p e r a t i n g at 22.49 M H z . T h e p r o d u c t of t h e ( 3 0 r e a c t i o n , g l u c o n o l a e t o n e , w a s
169
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i o I-::-0---! o-I Fig. 1. S c h e m e o f the apparatus for m e a s u r e m e n t o f the m e m b r a n e potential with the G O reaction in the silk fibroin membrane. (1) Right c o m p a r t m e n t , (2} left c o m p a r t m e n t , {3) salt bridge, (4) substrate solution, (5) G O i m m o b i l i z e d silk fibroin m e m b r a n e , (6} Stirrer.
measured in the aqueous solution, where the p H was adjusted with 0.1 M s o d i u m h y d r o x i d e a n d h y d r o c h l o r i c acid aqueous solutions. Moreover, the t3C N M R spectrum of the p r o d u c t in the silk fibroin m e m b r a n e after the G O reaction was observed directly. After the ( 3 0 immobilized m e m b r a n e was immersed in 0.5 M glucose aqueous solution ( p H 7) for 30 rain, the m e m b r a n e was picked up, a n d then measured. Measurement o f p H after the GO reaction T h e p H c h a n g e of the G O immobilized silk fibroin m e m b r a n e after the e n z y m e reaction was measured b y the following m e t h o d . T h e G O immobilized silk fibroin m e m b r a n e was d i p p e d in 0.1 m M p o t a s s i u m p h o s p h a t e buffer ( p H 7) overnight. T h e n the m e m b r a n e was a t t a c h e d to a glass p H electrode ( M o d e l TP-101, T o k o Chemical L a b o r a t o r i e s Co., T o k y o , J a p a n ) with a n y l o n net a n d an O-ring. A given a m o u n t of glucose solution (1 M) was injected into the reaction c h a m b e r with the assembled p H electrode. T h e n the p H c h a n g e was recorded. T h e change of p H in the silk fibroin m e m b r a n e was also m e a s u r e d with a p H indicator, 3-sulfo-2,6-dichloro-3",3"-dimethyl-4"-hydroxyfuchsone-5",5"-dicarboxylic acid, trisodium salt ( C h r o m a z u r o l S, D o j i n d o Laboratories, ~ , u m a m o t o , Japan), by m o n i t o r i n g the a b s o r b a n c e at 430 n m b y m e a n s of a n U V s p e c t r o m e t e r (U-3200, Hitachi) equipped with a t h e r m o s t a t e d cell holder. Determination of the f i x e d charge density o f the silk fibroin membrane . . . . . . T h e m e m b r a n e p o t e n t i a l was measured at a given p H b y c h a n g i n g the concentration of KC! in each c o m p a r t m e n t while m a i n t a i n i n g the c o n c e n t r a t i o n ratio of KCI
170
°im'n/
" .
.
.
.
.
Fig. 2. Typical response of the membrane potential with the reaction of 2% GO immobilized in the silk fibroin membrane after injection of glucose (final concentration = 1 raM). at 2:1. The fixed charge density was calculated p o t e n t i a l a s a f u n c t i o n o f p H [19].
using the observed
membrane
RESULTS AND DISCUSSION
M e m b r a n e potential induced by the G O reaction The membrane potential of the GO immobilized silk fibroin membrane during the enzyme reaction was measured. Figure 2 depicts a typical response of the membrane potential of the GO immobilized silk fibroin membrane after injection of t h e s u b s t r a t e . I n t h i s c a s e , t h e c o n c e n t r a t i o n o f p o t a s s i u m p h o s p h a t e in e a c h cell w a s t h e s a m e a n d d i d n o t c h a n g e d u r i n g t h e i n j e c t i o n . It h a s b e e n r e p o r t e d p r e v i o u s l y t h a t n o l e a k a g e o f G O f r o m t h e silk f i b r o i n m e m b r a n e w a s o b s e r v e d [5]. T h e m e m b r a n e p o t e n t i a l i n c r e a s e d g r a d u a l l y a n d r e a c h e d a p l a t e a u , as s h o w n in P i g . 2. I n t h e a b s e n c e o f G O , n o m e m b r a n e p o t e n t i a l w a s o b s e r v e d a f t e r t h e injection of glucose solution. Figure 3 shows a plot of A E, the membrane potential in t h e s t e a d y s t a t e , a g a i n s t t h e g l u c o s e c o n c e n t r a t i o n o f t h e s o l u t i o n in t h e l e f t compartment. The potential increased with increasing glucose concentration in the range of 0 to 5 mM. Previously, the membrane potential of an enzyme immobilized m e m b r a n e h a d b e e n m e a s u r e d w i t h a n a l b u m i n m e m b r a n e b e a t i n g u r e a s e [22] a n d
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Fig. 4. laC NMR spectra of products of the GO reaction. (31uconolactone aqueous solution at two pHs: (A) 6.8 and (B) 3.0. (C) Porous silk fibroin membrane after immobilized GO reaction. C1-C6: (31uconic acid, (A) gluconolactone. w i t h a c e t y l c h o l i n e s t e r a s e i m m o b i l i z e d ossein g e l a t i n m e m b r a n e o r a l b u m i n m e m b r a n e [181. T h e f o r m e r w a s m e a s u r e d w i t h d i f f e r e n t p h o s p h a t e b u f f e r c o n c e n t r a tions o n e a c h side o f t h e m e m b r a n e . T h e m e m b r a n e p o t e n t i a l c h a n g e s w e r e b a s e d o n t he r e c i p r o c a l e f f e c t b e t w e e n e n z y m e a c t i v i t y a n d t h e a p p e a r a n c e o f i o n s a f t e r t h e e n z y m e r e a c t i o n , i.e., a m o d i f i c a t i o n o f t h e local p H a n d a f e e d b a c k a c t i o n o f b u f f e r d i f f u s i o n f r o m t he o u t s i d e s o l u t i o n . T h e l a t t e r s t u d y also p o i n t e d o u t t h e e f f e c t o n t h e m e m b r a n e p o t e n t i a l o f i o n i c sp ecies p r o d u c e d b y t h e e n z y m e . H o w ever, it is i n s u f f i c i e n t to u n d e r s t a n d th e d e t a i l e d info~',~ation c o n c e r n i n g t h e effect of products on the enzyme support.
SaC N M R observation o f the GO reaction in the silk fibroin membrane It is n e c e s s a r y t o p a y a t t e n t i o n to th e i o n i c p r o d u c t , g l u c o n i c acid, b e c a u s e t h e i o n i c p r o d u c t in t h e m e m b r a n e m a y g e n e r a t e th e i o n d i f f u s i o n in th e m e m b r a n e a n d / o r t h e local p H c h a n g e in t h e m e m b r a n e . I n a d d i t i o n , t h e fine s t r u c t u r e o f t h e silk f i b r o i n m e m b r a n e is c o n s i d e r e d to b e a f f e c t e d b y t h e i o n i c p r o d u c t . H o w e v e r , th e s t a t e of t h e i o n i c species p r o d u c e d in t h e m e m b r a n e w a s n o t o b s e r v e d d i r e c t l y in spi t e o f t h e i m p o r t a n c e o f t h e i o n i c p r o d u c t . T h e r e f o r e , w e o b s e r v e d t h e s t a t e o f t h e p r o d u c t s a f t e r t h e e n z y m e r e a c t i o n in t h e silk f i b r o i n m e m b r a n e u s i n g ~aC N M R . F i g u r e s 4 A a n d B s h o w ~aC N M R s p e c t r a o f g l u c o n o l a c t o n e a q u e o u s s o l u t i o n at t w o p H s , 6.8 a n d 3.0, respectively. All p e a k i n t e n s i t i e s a t t r i b u t a b l e to g l u c o n i c a c i d a r e
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from gluconolactone, and of KCI. The concentration ratio (c2/c~) was changed from 1 to 2. (e) Gluconic acid (cl = 0.01 M), (o) gluconic acid (ca ----0.001 M). (zx) KCI (c i ----0.01 M). s t r o n g e r t h a n t h o s e of g l u c o n o l a c t o n e c a r b o n s e v e n at p H = 3.0. F u r t h e r , Fig. 4 C d e p i c t s the 13C N M R s p e c t r u m o f the p r o d u c t in p o r o u s G O i m m o b i l i z e d m e m b r a n e g e n e r a t e d by the i m m o b i l i z e d e n z y m e in t h e m e m b r a n e . T h e p e a k s can b e a s s i g n e d exclusively to the c a r b o n s o f g l u c o n i c acid, a l t h o u g h t h e p e a k s are b r o a d b e c a u s e o f the low m o b i l i t y o f t h e p r o d u c t s r a t h e r t h a n t h e high m o b i l i t y o f t h o s e in s o l u t i o n . U n d e r t h e s e c o n d i t i o n s , t h e c a r b o n s o f silk fibroin w e r e n o t o b s e r v e d . T h a s , it is c o n c l u d e d that g l u c o n o l a c t o n e , w h i c h is the p r o d u c t o f t h e G O r e a c t i o n , is r a p i d l y h y d r o l y z e d to g l u c o n i c acid in t h e silk fibroin m e m b r a n e . In a d d i t i o n , p H t i t r a t i o n o f g l u c o n i e acid s u g g e s t e d t h a t t h e p H in the G O i m m o b i l i z e d m e m b r a n e is s h i f t e d to the acidic side b y t h e e n z y m e reaction.
Effects of gluconic acid on the membrane potential of the silk fibroin membrane F i g u r e 5 d e p i c t s the c o n c e n t r a t i o n m e m b r a n e p o t e n t i a l o f the silk f i b r o i n m e m b r a n e w i t h g l u c o n i c acid p r o d u c e d f r o m g l u c o n o l a c t o n e . This i n d i c a t e s t h a t t h e a d d i t i o n o f g l u c o n i c acid to o n e side o f t h e silk f i b r o i n m e m b r a n e g e n e r a t e s a m e m b r a n e p o t e n t i a l a n d t h a t t h e p o l a r i t y is t h e s a m e as with KCl. H o w e v e r , t h e m e m b r a n e p o t e n t i a l d i f f e r e n c e s u s i n g g l u c o n i e acid are small c o m p a r e d w i t h t h o s e u s i n g KCI, s u g g e s t i n g an effect o f p H c h a n g e o n t h e m e m b r a n e p o t e n t i a l . W h e n t h e g l u c o n i c acid is p r e f e r e n t i a l l y p r o d u c e d w i t h t h e i m m o b i l i z e d G O r e a c t i o n at o n e side o f t h e m e m b r a n e , this i o n i c species m a y d i f f u s e across t h e silk f i b r o i n m e m b r a n e . It is e x p e c t e d that a p H c h a n g e o c c u r s locally, c a u s e d b y t h e p r e s e n c e o f g l u c o n i c acid. Such a p H c h a n g e o n t h e m e m b r a n e s u r f a c e a n d in t h e
173
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Fig. 6. Plots of the rate of pH change of the GO immobilized silk fibroin membrane against the concentration of glucose. (o) 0.2% GO, (A) 1.0% GO, (~) 2.0% GO.
silk f i b r o i n m e m b r a n e was m e a s u r e d with b o t h a p H glass e l e c t r o d e a n d U V m e t h o d s . T h e f o r m e r m e t h o d gives p H i n f o r m a t i o n c o n c e r n i n g t h e s u r f a c e n o t in c o n t a c t w i t h t h e s u b s t r a t e directly, w h i l e the l a t t e r o n e gives t h e i n f o r m a t i o n o f p H c h a n g e o n t h e o t h e r m e m b r a n e s u r f a c e a n d in t h e m e m b r a n e . F i g u r e 6 s h o w s the r e l a t i o n s h i p b e t w e e n t h e rate o f p H c h a n g e in the s t e a d y s t a t e a n d t h e g l u c o s e c o n c e n t r a t i o n in t h e r e a c t i o n c h a m b e r . D u r i n g t h e G O r e a c t i o n , t h e p H o n the m e m b r a n e surface n o t in c o n t a c t w i t h t h e s u b s t r a t e was s h i f t e d to t h e a c i d i c side a n d t h e rate of t h e p H c h a n g e d e p e n d e d o n t h e c o n c e n t r a t i o n o f g l u c o s e in t h e r e a c t i o n c h a m b e r . T h e p l o t s are r e g a r d e d as s t r a i g h t lines, t h e slopes o f w h i c h i n c r e a s e w i t h i n c r e a s i n g c o n t e n t o f i m m o b i l i z e d ( 3 0 . T h e s l o p e s are 0.33 x 10 - a , 4.17 x 10 - 3 a n d 12.3 x 10 - 3 A p H m i n -1 m M - t , for G O c o n t e n t s o f 0.2%, 1.0% a n d 2.0%, respectively, w h i c h c o r r e s p o n d to t h e a m o u n t o f g l u c o n i c acid p r o d u c e d b y t h e ( 3 0 r e a c t i o n in t h e silk f i b r o i n m e m b r a n e . T h e p H c h a n g e o n t h e m e m b r a n e s u r f a c e in c o n t a c t w i t h t h e s u b s t r a t e w a s also e s t i m a t e d , f r o m t h e U V m e t h o d . T h e a b s o r b a n c e o f a p H i n d i c a t o r , C h r o m a z u r o i S, in t h e p r e s e n c e o f b o t h a G O i m m o b i l i z e d m e m b r a n e a n d g l u c o s e was r e c o r d e d c o n t i n u o u s l y a n d t h e n t h e initial rate o f t h e p H c h a n g e w i t h t h e e n z y m e r e a c t i o n w a s d e t e r m i n e d b y u s i n g t h e c a l i b r a t i o n curve. F o r a G O c o n t e n t o f 0.2%, t h e rate w a s 9.8 x 10 - 3 A p H m i n -~ m M -1. T h u s , t h e p H c h a n g e at t h e surface in c o n t a c t w i t h t h e s u b s t a n c e w a s m u c h larger t h a n t h a t o b s e r v e d u s i n g t h e p H e l e c t r o d e . T h i s result i n d i c a t e s that the p r o d u c t i o n o f g l u c o n i c acid b y t h e i m m o b i l i z e d ( 3 0 r e a c t i o n m a y c h a n g e the p H o f b o t h t h e i n n e r a n d s u r f a c e r e g i o n s o f t h e m e m b r a n e a n d t h a t t h e c o n c e n t r a t i o n of p r o d u c t at b o t h surfaces o f t h e m e m b r a n e is n o t equal. T h e s e results agree w i t h the c o n c e n t r a t i o n m e m b r a n e p o t e n t i a l w i t h g l u c o n i c acid, t h e ~3C N M R o b s e r v a t i o n o f g l u c o n i c acid a n d also w i t h a t h e o r e t i c a l s t u d y o f t h e g e n e r a l t r a n s p o r t of t h e p r o d u c t s o f an e n z y m e m e m b r a n e [23]. T h e n o n - u n i f o r m d i s t r i b u t i o n o f t h e i o n i c species o v e r t h e t w o m e m b r a n e surfaces is c o n s i d e r e d t 0 r e s u l t n o t o n l y from i o n d i f f u s i o n b u t also f r o m t h e a s y m m e t r i c c h a n g e o f t h e m i c r o e n v i r o n m e n t of the silk f i b r o i n m e m b r a n e (see b e l o w ) .
174
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Fig. 7. Fixed charge density of various silk fibroin membranes as a function of pH. McIlvaine buffer concentration was 0.01 M. (o) GO immobilized silk fibroin membrane, (o) silk fibroin membrane.
T h e effective fixed c h a r g e d e n s i t y o f t h e silk fib ro in is e x p e c t e d to b e a f f e c t e d by t h e g l u c o n i c a c i d d u r i n g t h e e n z y m e r e a c t i o n in th e m e m b r a n e . F i g u r e 7 s h o w s p l o t s o f t h e fixed c h a r g e d e n s i t y as a f u n c t i o n of p H . T h e silk fib ro in m e m b r a n e h a s n e t positive c h a r g e s in t h e a c i d i c r e g i o n a n d n e g a t i v e o n e s in t h e b a s i c r e g i o n relative to t h e isoelectric p o i n t , w h i c h is p H 4.3 for th e o r i g i n a l silk fib ro in m e m b r a n e a n d p H 4.5 for the ( 3 0 i m m o b i l i z e d silk f i b r o i n m e m b r a n e . I n p a r t i c u l a r , t h e e f f e c t i v e fixed c h a r g e d e n s i t y c h a n g e d r e m a r k a b l y in t h e p H r a n g e f r o m 7 to t h e p H o f t h e isoe l e c t ri c p o i n t . T h e r e f o r e , this c h a n g e relates to t h e g e n e r a t i o n o f the D o n n a n p o t e n t i a l o n e a c h s u r f a c e of t h e ( 3 0 i m m o b i l i z e d silk f i b r o i n m e m b r a n e . In a d d i t i o n , t he fine s t r u c t u r e o f t he silk f i b r o i n m e m b r a n e is e x p e c t e d to b e i n f l u e n c e d b y t h e i n t e r a c t i o n b e t w e e n g l u c o n i c a c i d a n d t h e silk f i b r o i n e n z y m e s u p p o r t , s u g g e s t i n g a n effect o f m e m b r a n e s t r u c t u r e o n t h e m e m b r a n e p o t e n t i a l c o u p l e d to the immobilized enzyme reaction. I n g e n e r a l , t h e m e m b r a n e p o t e n t i a l d i f f e r e n c e w i t h a n artificial fixed c h a r g e m e m b r a n e s u c h as a silk f i b r o i n m e m b r a n e is e x p r e s s e d as fo llo w s [24]: A E AEDon.~ . + A E d i f f u s i o n , w h e r e A E D o . ~ ~ a n d AEdiffu~io n a r e th e D o n n a n a n d d i f f u s i o n p o t e n t i a l s , respectively. T h e f o r m e r p o t e n t i a l is t h e s u m o f t h e t w o s u r f a c e p o t e n tials at b o t h sides o f t he m e m b r a n e a n d t h e l a t t e r is t h e d i f f u s i o n p o t e n t i a l in t h e m e m b r a n e . T h e e n z y m e r e a c t i o n in t h e m e m b r a n e m a y c a u s e a c h a n g e in b o t h AEDo~na.~ a n d AEd{ff,,~io.. It is p a r t i c u l a r l y i m p o r t a n t to reveal t h e b e h a v i o r o f i o n i c spe c i e s a f t e r the e n z y m e r e a c t i o n in t h e silk fib ro in m e m b r a n e . It will t h e r e f o r e b e n e c e s s a r y t o i n v e s t i g a t e t h e m o b i l i t y o f ionic species in t h e silk f i b r o i n m e m b r a n e a n d the i n t e r a c t i o n b e t w e e n the p r o d u c t a n d th e silk fibro.ln in o r d e r to i m p r o v e t h e r e s p o n s e a n d to d e s i g n a n effective s t r u c t u r e for th e silk f i b r o i n m e m b r a n e as a n enzyme support. •. . . . .
175 REFERENCES 1 L. Grasset, D. Cordier and A. Ville, Biotechnol. Bioeng., 19 (1977) 611. 2 L. Grasset, D. Cordier and A. Ville, Process Biochem., 14 (1979) 2. 3 S. Miyairi, M. Sugiura and F. Fukui, Agric. Biol. Chem., 42 (1978) 1661. 4 D. Cordier, R. Couturier, L. Grasset and A. Viile, Enzyme Microb. Technoi.o ,5 (1982) 249. 5 A. Kuzuhara, T. Asakura, R. T o m o d a and T. Matsunaga, J.Biotechnoi., 5 (1987) 199. 6 T. Asakura, Bioindustry, 4 (1987) 36. 7 T. Asakura, H. Yoshimizu, A. Kuzuhara and T. Matsunaga, J. Seric. Sci. Jpn., 57 (1988) 203. 8 T. Asakura, J. Kanetake, and M. Demura, J. Polym. Plast. Tecknoi. Eng., 28 (1989) 453. 9 M. Demura and T. Asakura, Biotechnol. Biocng., 33 (1989) 598. 10 M. Demura, T. Asakura, E. Nakamura and H. Tamura, J. Biotechnol., 10 (1989) 113. 11 M, Demura, T. Asakura and T. Kuroo, Biosensors, ,5 (~989) 361. 12 H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (I990) 127. 13 H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 1745. 14 T. Asakura, H. Yoshimizu and M. Kakizaki, Biotechnol. Bioeng., 35 (1990) 511. 15 R. To r and A. Freeman, Anal. Chem., 58 (1979) 1042. 16 T. Shinbo, M. Sugiura and N. Kamo, Anal. Chem., 51 (1979) 100. 17 R.H. William and H.B. Halsall, Anal. Chem., 57 (1985) 1321A. 18 A. Friboulet and D. Thomas, Biophys. Chem., 16 (1982) 153. 19 M. Sugiura, Nippon Nogei Kagaku Kaishi, 47 (1973) 563. 20 M. Demura, A, Kitamura, A. Shibamoto and T. Asakura, J. Appl. Polym. Sci., 36 (1988) 535. 21 T. Asakura, Y. Watanabe, A. Uchida and H. Minagawa, Macromolecules, 17 (1984) 1075. 22 A. David, M. Metayer, D. Thomas and G. Broum, J. Membrane Biol., 18 (1974) 113. 23 T. Ciftci and W.R. Vieth, J. Mol. Catal., 8 (1980) 455. 24 Y. Kobatakeo K. K.urihara N. K am o in T. Takamura and A. Kozawa (Eds.), Surface Electrochemistry. Advances and Concepts, Japan Scientific Societies Press, Tokyo, 1978, p. 1.