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Direct Electrochemical Coupling of Components of the Biological Electron Transfer Chain to Modified Surfaces Molecular Recognition between Cytochrome c Peroxidase and Cytochrome c.

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ble to a rapid thiono-thiolo rearrangement supported by the
/{-silicon effect with formation of 0,O.S-trialkylphosphorothioates 6a-r. which. under the deprotection conditions. form
dialkylphosphates 8a-c. The properties of the 2-(diphenylmethyl)silylethyl group in Sd, however, are compatible with
oligopliosphorothioate synthesis, that is, the thiono - thiolo rearrangement driven by the lj-silicon effect is largely suppressed
under acidic its well as under basic conditions. while selective
deprotection under miid conditions through a p-fragmentation
mechanism is preserved.
2 a d : All opcrntions wcreperformed under exclurionofmoisture(Ar atmosphere).
To :I c d d (ice hntli) ~ o l u t i o nof PCI, (27.5 g. 200 nimol) in dry Et,O (I00 mL) was
added dropwire 'I wlution of the /j-silylethanol derivatives la d (50 mmol) in dry
Et,O ( 5 0 inL) 1 hc ICC heth w a s removed, and after 2 h the solution was concentrated i n v;icuo (70 1oi-I. then I T o r ) . Dry Et,O (200mL) \\\:asadded. followed by
drop\\\:isciidditioii 01 f+YiPr2 ( 5 0 inL). After stirring for 24 h a t room tenipei-ature
the mixture \\:I\ Iiltcred. a n d the solvent removed in vacuo to afford 2 a - d as
colorless o i l s i n iiciii- quantitative yield. "P N M R (81 MHz. CDCI,): d =123.7
( 2 a ) . 1234 (Zb) 123.9 ( 2 c ) . 123.4(Zd).
3 a d: C!ndt~ cxcIusion ofmoisture. IH-tetrarole (12 mmol) was added to a stirred
solutioii < ) I Z a d ( 3 0 mind) i n dry C H , C N (600 mL). After 20 min this solution
iddcil to S ' ~ ~ ~ - ( J . 4 ' - d i n 1 e t h o x y t r i t ~ l ) t h y m i (1
d i 5n emmol) within 30 min. Al'ter
1. t i i ~ t h y l a i i i i i i c(30 m L j uas Lidded. and tlie solution %;is stored LLI
-20 C for
17 h Aftcr i'iltratioii tlie solution \\\:as concentrated i n vacuo. The oily residue was
I xctate. and the solution extracted with NaHCO, ( 1 M) and H,O.
c.wiisdr-ied (N;i,SO,).coiicenrrated. and the remainingoil puritied
h) flash ~liroiiiat~~gi-,iph?
o n zilica (eluent. ii-hexanes;ethyl acetate:NEt, 90:lO~O.l
to 5 0 5 O ' O . I ) t o furnish 3 a d. "P N M R (81 MHz, CDCI,): d = 145.9. 146.1 ( 3 a ) .
I45 X. 146.0 (3h). l4j.Y. 146.1 (3c). 146.0. 146.3 (3d).
4a d Uiidcr exclurion of moisture I I/-tetrazole (6 mniol) was added to a stirred
s o l u t i o n of 3 a d ( 2 mind) and 3'-O-acetylthymidine ( I minolj in dry CH,CN
(10 m L l Aftei- 2 11 ii mlution of31/-1,2-beii~odithioI-3-one-l.l-dioxide
( 5 mmol) in
dry C'H ,C'N ( 5 m L ) \+;is Lidded. After 30 min trtethylamiiie (1 mL) wils added. and
the mixt~irefiltered. The filtrate was concentrated in vaciio. The residue was dissol\cd i n ethql iicctiite. and the solution extracted with aqucous NaHCO, ( 0 . 5 ~ )
;ind H 2 0 . Thc orpii>ic phase was dried (Na,SO,). concentrated. and the residue
purilicd ti! 11:i\Ii i l i i oniatogl-aphy on silica (eluent: ti-hexanes,ethyl acetnte,.NEt,
5 0 : 5 0 0.1 t o 10 X 0 , O . l ) to furnish 4a-d."P N M R ( X I MH7. CDCI,): 6 = 67.5.
67.6 (4a).(17 6. 67.7 (4h). 67.2. 67.5 (4c).67.4. 67.5 (4d).
Received' May 3. 1995 [Z79531E]
German version: Airgoii'. Cheiir. 1995. 107. 2584-25x7
Keywords: 'intisense systems . neighboring-group effects
nucleotides . phosphorus compounds . rearrangements
Direct Electrochemical Coupling of Components
of the Biological Electron Transfer Chain to
Modified Surfaces : Molecular Recognition
between Cytochrome c Peroxidase and
Cytochrome c**
Li Jiang, Calum J. McNeil, and Jonathan M. Cooper*
The modification of surfaces with proteins is established as a
method for preparing analytical devices capable of biomolecular recognition. For example, research on ligand binding at interfaces has focused on the immobilization of both antibodyantigen and avidin-biotin coup1es.l" 21 resulting in commercial
biosensing methodsr3] as well as in techniques for controlling
Likewise, the immobilization of enzymes at electrodes has enabled the study of heterogeneous biocatalysis. and has produced an attractive sensing technology
through the indirerl measurement of either a cofactor associated
with the biochemical a ~ t i v i t y , ' or
~ ' of an immobilized inorganic
redox mediator in electrochemical communication with a
Over the last decade. there has been a large body of literature
exploring direct electron transfer (ET) between immobilized
proteins and conducting surfaces. To date, however, research
has been limited to small model proteins,['.'' and it is now
known that suitable modification of an electrode provides an
appropriate environment for studying Facile, quasi-reversible
bioelectrochemistry. A particular example involves using a film
of self-assembled co-mercaptocarboxylic acid on gold to measure ET kinetics of cytochromec (cyt c) across ;I "zero-length"
carbodiimide coupling.[71These architectures have proved exci ting for those involved in biosensor research. as they offer the
possibility of electrochemically "wiring" larger proteins to electrodes without ET mediators.
We now present a development of these ideas to produce more
complex assemblies at which biomolecular recognition between
redox proteins can be studied. We show the immobilization of
components of the biological ET chain and demonstrate the
direct electron transfer between the modified gold electrode and
the immobilized heme proteins. We also measure the enhanced
electrocatalytic response due to heterogeneous biomolecular
recognition at the protein-modified surface.
Cytochromec peroxidase (CcP. [EC 1 .I 1.1.51) is a heme-containing enzyme (35 kDa) that acts as an alternative terminal
electron acceptor (complex Iv) in anaerobic ycdst (&rcchuromyccJsc.errvisiue). Its interaction with cyt c (1 2.6 kDa). another
heme protein, has provided a model upon which an understanding of protein-protein ET has been established.[*] CcP
reacts rapidly with H,O, to form a doubly oxidized form of the
enzyme containing a bound radical (compound 11). This intermediate compound reacts further with two molecules of reduced
cytc (cytc,,,) to regenerate the reduced form of CcP (Fe"').
Long range ET between the two heme centers occurs across the
cyt c-CcP complex, itself stabilized by ion-pairing between
lysine groups in cytc and a ring of acidic amino acid residues
[ I ] S T C'rookc 111 Uurgw:\ .Miw/iciiiu/ Ciimirsiui. wrd Drug fid. / (Ed.:
M E. Wolff). Wilcy. 1995. pp. 863 900 and references therein.
121 S. T ('rookc. A i r i i i i d ;!4rcrbig of //re Fizikwrioir of th? Aiiirriwir Socii,i~.( I /
E \ p i , r i i w i i / d U/o/o,yi. ( F A S E B j , April 1994. Anaheim. CA, USA.
13) A lhom~>,-thymidine
dialkylphosphate dodecainer (T,?) w a s synthesized on
('PG u\ing 1-(diphenylmethylsilyl)ethylphosphorainidite
(3d) with an average
cciiipliiig cl'ficiency 01' 9X 5 ' X . a j V. T. Ravikumar. H. Sasmor. D. L. Cole,
Ui(iory . W d (%cni. Lcrr. 1993. 3, 2637 2640. h) V. T. Ravikuiixir. T. K
N'yrrykienici. 1). L. Cole. T<erra/w.r/roti1994.50. 9255- 9266.
141 A hi)i~i~i-thyiiiidine
phosphorothioate hcptamer (T.) was synthcsiLed on CPG
u\lng 3d w i t h i i i i avcnige couplnig efficiency of 9 9 % . V. T.Ravikumar. D. L.
i'olr.. Gcirv 1994. 149. 157 -161.
151 S L Beauc,igc. M . H . Caruthers. %.rru/ici/roii Lerr. 1981. 22. 1859 1862.
[h] N I). Sinha. J. Biernzit. H. Kiister. Erru/rrr/r.on Lcrt. 1983. 24, 5843 5846.
[7] S. Honda. 7 Hata. 7i,rriihr[/ron L c z r r . 1981. I?. 2093-2096.
[XI J. A. Soderqmt. I Rivera. A. Negron, J. Orfi. Cheiri. 1989.54. 4051 -4055.
191 R. 1'. lqcr. 1. R . Phillips. W. Egan. J. B. Regan. S. L. Beaucage. J. Org. C/ioin.
1990. 33. 4643 4699
[ 101 a ) t l . Tcichiiiiinn. G . Hilgetag. Airgew. C/toii. 1967. 7Y. 1077- 1088. A ~ g c i t . .
[*] Dr. J. M Cooper, Dr. L. Jiang
C / i m i . / / I / . Ld Eii,?/. 1967. 6. 1013 1023: b j R. G. Cooks. A . F. Gerrard, J.
Department of Electronics and Electrical Engineering
C/iuii ,Sot / j 1968. 1327 1333, c j K . Brurik. N'.1. Stec. J. Org. C h r r . 1981. 46.
University of Glasgow. Glasgow GI?XLT ( U K )
1625 16311. ( d ) C. D. Poulter. D. S. Mautz. J. Aiii. Ciicm. So.. 1991. 113.
Telefax: I n t . code + (141)330-4907
4x95 4003
e-mail: jnicooperra
[ I l l .I. B L.imbeit. f i , r r d i d r o i i 1990. 46. 2677 -2689 and references therein.
Dr. C J. McNeil
[I?]N SIiimi/ii. S Watannhe. F. H;i)ak;i\\\:a,S. Yasuhara. Y, Tsuno. T, l n u u . Bid/,
Department oi' Clinical Biochemistr)
( /I~.III.S o r . .lpr. 1994. 157. 500- 504.
Univcraity of Newcastle upon Tyne ( U K )
[I31 I n :I control experiment u e deprolected 6c under the same conditions. Com] i o u n d 8 c ~ . i ~ t h c e x c l u s i \ ~ e p r o d u( c" tP N M R i i =0.02). h , = 1 . 4 ~ 1 0 ~ ' s ~ ' [**I We thank Prof. J. E. Erman. Northern Illinois Universit). USA. for the original
( I , I = 13.4 11)
sample of CcP. and EPSRC (Grant GRiJI0189) for support.
around the heme edge of CcP.[*l The study of cytc-CcP is of
particular interest to us as it contains pathways along which
electrons flow as a direct result of the interaction between the
two proteins.
The proteins were immobilized using 3,3'-dithiobissulfosuccinimidyl propionate (DTSSP). DTSSP assembles spontaneously on a bare gold electrode with a surface coverage. estimated by
integration of its oxidation peak at 150 mV vs. Ag/AgCl, of
1.5 x 1 0 - l o m o l cm-'. In order to study the cytc-CcP interaction electrochemically. in the first instance, cyt c was immobilized in a single-step reagentless procedure involving the reaction of its (surface) lysines with DTSSP. The biomolecular film
assembles at a gold surface, providing electrochemical access to
the heme redox center of cytc. apparent in deoxygenated electrolyte (the experimentally measured half wave potential
El,, = -153 mV vs Ag/AgCl, AEp =70 mV, L' = I 0 mVs-',
Fig. 1 ) . Integration of the reduction current of the immobilized
cytc indicated that the surface concentration was 4.1 x
lo-" molcm-'. This value is generally in agreement with those
obtained by others who have immobilized proteins at selfassembled mono layer^.'^]
Fig. 2 . Top: Scheme of the electrocatalytic reactions for the reduction of immobilized cytc with enzymatic regeneration of cytc,,,,, by solution phase CcP in the
presence of H , 0 2 . Bottom: DC cyclic voltammograms of immohilized cytc (vs.
AgiAgCI electrode): a) in 0.1 moldm-' sodium phosphate buffer, pH6.3: h) on
addition o f 3 0 p ~H,O,, and c)-f) in the presence of successive additions of 0.43.
0.86, 1.29, and 1 . 7 2 CcP,
~ ~ respectively ( L = 5 mVs-'). The reduction current for
immobilized cytc is enhanced, because the inter-protein ET rate is about 1000 s - I ,
which is nearly four orders of magnitude faster than that at the electrode surface [16]. El. =electrode.
E[mV]Fig. 1 . DC cyclic voltammogram ( I ! = 10 mVsC') depicting the direct electrochemin a degassed solution
istry of immobilized cytc fAu\DTSSP,cytc -)
(0.1 moldm-3 sodium phosphate buffer, pH6.3). Also shown are control experiments: the DTSSP monolayer alone (Au\,DTSSP . . . ).and Au\DTSSP\,;apo-cytc i n
the presence of 0 . 5 CcP
~ ~(--- -).
Cytc has eighteen surface lysine groups able to bind to
DTSSP. The ET route within the biomolecular film can be considered as a statistically averaged pathway, depending upon the
probability of binding of particular primary amines on the
protein surface. A weighted rate constant k& for ET between
the protein and the electrode was estimated with Laviron's model as 0.18 s- .[91 The linear dependence of the peak currents on
the potential scan rate, measured between 10 and 50 mVs-'
(data not shown), indicated that cytc exists as a surfacebound species, [for example, for i,, y(/nA) = 9.7x(/mVs-') +
0.8 (/nA)]. This fact was further confirmed by the persistence of
the protein redox signal after exhaustive washing.
Upon addition of H,O, to the Au-cytc electrode, the reduction current is enhanced, consistent with previously characterized electrochemical reactions involving the reduction of hydroperoxide (Fig. 2b) .[''I
Subsequently, additions of CcP
(Fig. 2c-f) resulted in substantial increases in the reduction
currents due to enzymatic regeneration of immobilized cyt c,oxj
and its electrocatalytic reduction at the electrode surface. The
reaction scheme is illustrated in Figure 2.
The same Au-cytc assembly was also used to study proteinprotein recognition in the absence of H,O,. To this end, ET
reactions between soluble CcP,,,, and the modified electrode
were measured (Fig. 3 ) . As expected from its biological function, and due to the relatively free accessibility of the immobilized heme redox center, CcP serves to regenerate immobilized
cytc,,,, (via compound I), resulting in an enhanced reduction
current at the electrode. as illustrated schematically in Figure 3.
In a series of complimentary experiments, CcP was immobilized using DTSSP, and its direct electrochemistry was characterized in deoxygenated electrolyte (Fig. 4). The weighted k'&
was calculated as 0.04 s - ' . The lower value for the ET rate
constant for CcP (with respect to cytc) is attributable to the
longer probable ET pathway between the heme and the gold
surface. Assignment of such a pathlength is, however, difficult
as there are 23 lysines on the surface of CcP, available to bind
with DTSSP.
Plots of ips,or ipc vs. 1: were linear [for example, for iTd,
y (/nA) = 9.1 x(/niVs-')
2.7 (/nA), data not shown], thus
confirming that the electrochemical reactions were confined to
the surface. The E',/, for immobilized CcP was estimated as
-254 mV vs. Ag/AgCl at 1: = 10 mVs-', relative to the reduction potential of CcP in solution (compound I1 containing Fe"'
ions). estimated as 848 mV vs. Ag/AgCI.["' This large shift in
the enzyme's redox character is attributed to the substantial
change in the local physical and chemical environment of the
immobilized heme redox center. Integration of the reduction
peak of CcP demonstrated that the surface coverage of the enzyme was 2.8 x lo-" molcm-2, a lower apparent value than
that measured for cytc.
On addition of soluble cytc to the Au-CcP electrode, the CcP
reduction current is enhanced (Fig. 4). The redox conditions
within the electrochemical cell are such that the reaction is reversed from that in nature. where CcP acts as a terminal electron
acceptor during tlie oxidation of cytc,,,,, ( E l , *cytc = + 40 mV
vs. AgjAgCI) .I'21 This reverse reaction has bccn studied, for
example by means of laser radiolysis.[*]
Previously. solution-phase components of the ET chain have
been shown to communicate with monolayers a t an electrode
surface.['3 ' 51 The novel electrode arrangement that we describe here for the electrochemical investigation of iminobilized
components of the ET chain offers potential advantages over
such homogenous models. Not only does the system mimic that
of nature, where different protein interactions occur at different
phase interhces. but from an experimental viewpoint, problems
of solution resistance are negligible. Two aspects of the study are
of particular interest: the work provides an example of direct
faradaic signal transduction due to protein recognition at a
protein modified surface: in addition, the techniques used can to
manipulate the redox character of biological molecules through
Esperimcntcil Prorcdurr
E[rnV]Fig 3 Trip Scheme o f the electrocatalytic reaction showing the one-electron enzyiniitic onid;ition ol~iininobilizedcytc b y solution phase CcP. with subsequent electrochcmic;il reduction. Bottom. D C cyclic w l t a m m o g r a m s of immobilized cyt c ( a ) .
enh:inced by tlie \uccessive addition of 0 . 3 0 (b).
~ ~ 0 . 6 0 p ~(c), 0 . 9 0 (d).
~ ~ I.XpM
( e ) , and 2 . 4 p i (1'1 CcP. i n the absence o f H,O, ( 1 , = 5 mVsC', all the other conditions <I\ in I'ig 2 )
H o r x heart cytc and CcP was immobili~edby mixing 10 mg iniL of lhe protein
within a n excess of DTSSP (IOmM solution in l O ( l m ~sodium phosphate buffer.
pH6.3) for 30 min. Alternatively. DSP was uscd (10 mM aolutioii in dimethylsulloxidc. diluted 1 5 with water) A gold electrode ( A = 0.031 em'. p o h h c d with
0.05 pm iilnmin3. rrcated with 2 5 % H,O, in H,SO,. and sonicated i n water for
1.5 minl was incubated in the enzyme cross-linker solt~~ion
oyernight iit 4 C . All
electrochemical meacurenients were made with ii BAS CV-37 ]piitentiortat and w'ere
collected either a s digital o r analog data set\. D C cyclic ~oltiiiiiinetricexperiments
were performed in a scaled two-chamber electrolytic cell. which had ii working
solution volume of 500 pL.
The direct electrochemical measurements
011 protcin werc carried oul i n
0 1 m o l d m ~ ' ~ o d i u iphosphatc
buffer. p H 6 3. which w a s dcoiygcnatcd Inr I S miii
~ i t nitrogen
prior to measurement The solution h a s niaintaiiicd under i i nitrogen
blanket during the experiment. Likc\\isc. all \tack solutions of reagents. prior to
;iddition to the electrochemical cell. were treated in a ~iinil;irI,ishion
Exclusion of dioxygen froin degassed solutions h a s confirined independently with
unmodified gold electrode. poised at -300 mV vs. Ag.AgC'I Control experimcnts i n nhich the heme redox center o f t h e proteins \here rcniovcd ( h y i n c u b a ~ i n g
the electrode in 4 . 0guanidine.HCL
pH 6.0 overiiight folloned by w'ishing) wcrc
carried nut for both electrodes. and are shown for cyt c in Figure 1
R e c c i ~ d April
2X. 1995
Revised version: August 14. 1995 177939IEj
German version: AN#CII. Chriii. 1995. 107. 2610 2613
1 lOOnA
Keywords: bioelectrochemistry * cytochromec peroxidase
electron transfer * immobilization
[ l ] H . Ebato. J. N . Herron. W Muller. Y. 0. K a h a t a . H . liingsdorf. P. Suci.
A i i p r . Chiwi. 1992. 104. 1064 1067: Aii,fyw. Chivi. I n r . Ed. Eii#/. 1992. 31.
10x7 1090.
[2] D. J. Pritchard. H. Morgan. J. M. Cooper. Aiigcii-. ( ' / i r w 1995. 107. 84-86.
A i i ~ r wC . h ~ m I. n / E d &q/. 1995. 34. 91 93.
[i] D. Altshuh. M. C . Dubs. E. Weiss. G. Zederlut7. M H . V Vanregeninoi-tcl.
Bioclicniisrri. 1992. 31. 6298 6304.
[4] L . C. Clark, C. Lyons. Aim. N Y A < u d . &I. 1962. 102. 79 41.
[5] I.Willner. N . Lnpidot. A. Riklin. R. Kasher. E Zahav). 1,. K a t ~J.. A n i . Cliiwi.
Soc. 1994. //6.142X ~ 1 4 4 1
[6] J. M. Cooper. K Greenough. C . J. McNeil. J Elwlrmiiid C , / i r w i . 1993. 347.
267 275
[7] M. Colliiiaon, E. F Bowden. M. J. Tarlov. Loiigtiltrrr 1992. S. 1247- 1250.
[8] E. Cheung. K . Tdylor, J. A. Kornhlatt. A. M Engli4i. (;. Llclxndon.
A . Miller. Pror.. .Viir. A r z d S1.r. USA 1986. X1. 1330 1333
191 E. Laviron. J. E l c r ~ ~ i - o i iChoii.
1979. 101. 1 Y -28.
[I01 J. Wilshire. D. T. Sawyer, A i c . Climi. /<C.V. 1979. I?. 105 110.
[ 1 I ] W L Purcell. 1. E. Erman, J. Ain. C'lirin Sor . 1976. YH. XI33 70.37.
[IZ] D. C . Rees. P r o i . N i r r . Ai.ud. S I I .USA 1985. 82, 3082 30x5.
[ I 3 1 F. A . Armstrong. A. M . Lannon. .I .Am C h n . Sor. 1987. 109. I21 1 7212.
[I41 M. Lion-Dagan. E. Katr. I Willner. J. Chivii. S i r . ( ' h o i i . < o i i i i i i i n i . 1994. 2741.
r i ~ i ~ 29,
[ l j ] S Baghy, P. D. Barker. L. H Guo. H. A. 0. Hill. B I ~/ i ~ ~Ci i i ~ . \1990.
3213 3719.
1161 A. F. Corin. R. A . Hake. G McLendon, J. T. Hazzard. C; rollin. B i o r ~ / i i 2 i i i i \ r r i
Fig. 4. Top: Scheme showing the one-electron enzymatic oxidation of immobilized
CcP by w l i i ~ i o nphase cytc. with subsequent electrochemical reduction. Bottom
DC cyclic voltamiiiogr;ims (1: = S m V s - ' ) of immobilized CcP in 0.1 moldn1C3
sod~iiiiiphosphate hutfer. p H 6.3 ( a ) and arrer successive additions of 7 . 0 (b)
~ and
4?pM C y l C ( C )
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molecular, chains, couplings, direct, components, biological, surface, electro, recognition, cytochrome, transfer, modified, electrochemically, peroxidase
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