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Archives of Biochemistry and BiophysicsVol. 362, No. 1, February 1, pp. 87–93, 1999Article ID abbi.1998.0981, available online at http://www.idealibrary.com on

he Optical Biosensor Studies on the Role of Hydrophobicails of NADPH-Cytochrome P450 Reductase andytochromes P450 2B4 and b5 upon Productive Complexormation within a Monomeric Reconstituted System

uri D. Ivanov,1 Irina P. Kanaeva, Vadim Yu. Kuznetsov, Michael Lehnerer,* Johannes Schulze,*eter Hlavica,* and Alexander I. Archakov

nstitute of Biomedical Chemistry RAMS, Moscow 119832, Russia; and *Walther-Straub Institute of Pharmacologynd Toxicology, Munich, Germany

eceived April 10, 1998, and in revised form September 10, 1998

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The optical biosensor study of interaction betweenicrosomal proteins—NADPH-cytochrome P450 re-

uctase, cytochrome P450 2B4, and cytochrome b5—as carried out in the monomeric reconstituted sys-

em in the absence of phospholipids. The formation ofndividual complexes was kinetically characterizednd their association and dissociation rate constantsere determined. The association rate constants for

he complexes formed were found to be close to theiffusiion limit—(0.5–4) 3 106 M21 s21—while their dis-ociation rate constants did not exceed 0.5 s21. It washown that the interprotein electron transfer can oc-ur both through complex formation and due to ran-om collision. The dominant role of hydrophobic mem-raneous protein fragments in formation of produc-ive electron transfer complexes was demonstrated.1999 Academic Press

Key Words: cytochromes P450 2B4 and b5; NADPH-ytochrome P450 reductase; kinetic constants; pro-uctive complexes; optical biosensor.

The cytochromes P450-containing monooxyganaseystems play an important role in the oxidation ofrugs, toxins, carcinogens, mutagens, and other xeno-iotics (1). It is known that cytochromes P450 (P450)unction by interacting with their redox partners,ADPH-cytochrome P450 reductase and cytochrome5. In recent years, P450 interaction with its protein

1 To whom correspondence should be addressed at Institute of

iomedical Chemistry RAMS, Pogodinskaya St. 10, Moscow 119832,ussia. Fax: 7 095 245 08 57. E-mail: yuiv@ibmh.msk.su.

ap

003-9861/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

artners has been the subject of many communica-ions. In some studies, using the spin equilibrium shiftnd fluorescence quench methods the complexes’ affin-ties and kinetic constants were determined (2–5). Inther studies, rate constants for interprotein electronransfer were reported (2, 3, 6–10). Presently the ever-idening application in molecular interaction research

s finding the optical biosensor method whereby theeal-time molecular interactions are explored withoutse of labeled molecules (11–14). In particular, theethod was effectively employed in research on com-

lex formation kinetics of water-soluble molecules (15).ur optical biosensor-based studies were undertaken

o determine the kinetic constants for the membraneroteins’ complex formation and decay. Since the na-ive environment of these proteins constitutes a phos-holipid bilayer membrane to which they are anchored,he kinetic rate constants are difficult to measure in itsresence. To obviate this difficulty, a soluble monoox-genase system containing hemo- and flavoproteinonomers (MRS)2 was reconstituted without a phos-

holipid membrane (16). To elucidate the mechanismshereby the system is operated, it was necessary toetermine the association and dissociation rate con-tants for complexes formed by redox partners and toompare the constants obtained with the appropriateate constants for electron transfer and hydroxylation.

2 Abbreviations used: PBS/t, potassium/Tween buffer; Fp, NADPH-ytochrome P450 reductase; 2B4, cytochrome P450 2B4; b5, cyto-hrome b5; d- or t-, full-length or truncated proteins, respectively;RS, monomeric reconstituted system; EDC, 1-ethyl-3-(3-dimethyl-

minopropyl)carbodiimide; NHS, N-hydroxysuccinimide; SOD, su-eroxide dismutase; im, immobilized.

87

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88 IVANOV ET AL.

edox partner interactions are accompanied by theppearance of their productive or nonproductive com-lexes—with intermolecular electron transfer eitherccurring or not occurring, respectively. Employing theptical biosensor method, it was possible to measurehe overall number of complexes formed—both produc-ive and nonproductive. In order for the electron trans-er to be efficient, the life-time of a bimolecular complexust exceed its characteristic electron transfer time.omparison of kinetic parameters obtained by the op-

ical biosensor method as employed in this study withlectron transfer rate constants obtained in hydroxyla-ion reactions enabled us to estimate the proportion ofroductive complexes in reference to the overall num-er of complexes formed.Full-length enzymes—cytochrome b5 (d-b5), NADPH

ytochrome P450 reductase (d-Fp), and cytochrome450 2B4 (d-2B4)—are amphipatic proteins containingydrophobic membrane fragments capable of incorpo-ating into the lipid bilayer. These fragments may beemoved by tryptic digestion—to obtain truncated pro-eins t-b5 and t-Fp (17)—or by gene expression—totimulate t-2B4 production (18). Based on comparisonf the biosensor-measured complex formation parame-ers for the full-length and truncated forms of 2B4, b5,nd Fp, the dominant role of hydrophobic membranousails in productive complex formation was demon-trated.

XPERIMENTAL PROCEDURES

Chemicals. Glucose oxidase, xanthine, xanthine oxidase, andatalase were purchased from Serva (Heidelberg, Germany). Super-xide dismutase and NADPH were obtained from Boehringer-Man-eim (Wien, Austria). Emulgen 913 was purchased from Kao AtlasOsaka, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidend N-hydroxysuccinimide were obtained from Fisons (UK). Otherhemicals were purchased from Reakhim (Moscow, Russia).Preparation of proteins. The proteins d-2B4, d-Fp, and d-b5 were

repared as described earlier (19–21). Specific content of d-Fp was3–13.5 nmol Fp/mg protein and its specific activity at 30°C was0–43 mmol of cytochrome c/min/mg of protein. Specific content of-2B4 was 17–18 nmol/mg of protein, A 276–417 5 0.5–1. Specificontent of b5 was 48–52 nmol/mg of protein, A 276–413 5 0.6. Purifiedryptic fragments of b5 and Fp were obtained from rabbit livericrosomes following their treatment with trypsin (17). The proteins

howed a single band on SDS–PAGE. Truncated cytochrome P450t-2B4) lacking N-terminal residues 2–27 was expressed in Esche-ichia coli and purified to apparent homogeneity as described else-here (18). The specific content of t-2B4 was 15 nmol/mg of protein.Monomerization of aggregates of the isolated Fp , 2B4, and b5 was

arried out as described previously (16) except that in the case of b5he 500 mM—instead of 100 mM—K phosphate (KP) buffer wassed.The determination of proteins’ binding parameters by optical bio-

ensor. The “resonant mirror” biosensor method is direct; i.e., itermits immediate real-time measurement of the overall number ofomplexes formed. The principle and instrumentation of the reso-ant mirror sensor have been described elsewhere (11–14). Determi-

ation of redox partners’ binding parameters within MRS was car-ied out in the IAsys biosensor cell (Fisons, UK) by covalently im-

m1

obilizing one of the partners on the carboxymethylated dextran-oated cuvette, using standard coupling chemistry methods. Thehemistry involved coupling of ligand amino groups to the carboxylroups on the dextran via 1-ethyl-3-(3-dimethylaminopropyl)carbo-iimide (EDC)/N-hydroxysuccinimide (NHS) (11–13). The sampleell (200 ml) was brought to thermal equilibrium and incubated at5°C in potassium/Tween buffer (PBS/t, 10 mM Na phosphate buffer,H 7.4, 138 mM NaCI, 2.7 mM KCI, 0.05% Tween 20) for 30 min.fter the activation with a mixture of EDC and NHS, protein immo-ilization was conducted for 15 min in 10 mM acetate buffer at pH.5, 4.5, and 4 for 2B4, Fp, and b5, respectively. The uncoupledigand was removed with PBS/t buffer. The uncoupled carboxylroups on the dextran were deactivated with 1 M ethanolamine, pH.5, and the sample cell was washed with PBS/t. The molecularurface concentrations of d- and t-2B4, d- and t-Fp, d- and t-b5 ofmmobilized (im) proteins were estimated to be (26 6 3) 3 10214,6.0 6 0.6) 3 10214, (10 6 1) 3 10214, (4.0 6 0.4) 3 10214, (43 6 5) 30214, and (14 6 2) 3 10214 mol/mm2, respectively. The binding ofmmobilized proteins with redox partners was followed by addingigand in large excess over the immobilized ligate after washing withP buffer. The association (k on) and dissociation (k off) rate constantsere determined under pseudo-first-order reaction conditions. Datatting was performed using the FASTfit program (Fisons) (14) basedn the equation

R 5 R0 1 R1 3 ~1 2 exp~ f 3 t!!, [1]

here R is the response of the device, t is the time period, R 0 is theesponse at t 5 0,

f 5 kon 3 c 1 koff, [2]

here c is ligand concentration, and R 1 is the response at t 5 } (athe equilibrium state)—relative to the response at t 5 0. The asso-iation and dissociation rate rate constants were calculated fromqs. [1] and [2]. The equilibrium constant (K eq), was calculated as

Keq 5 kon /koff, [3]

nd the complex life time was determined as

t 5 1/koff. [4]

The binding curve did not depend on the stirrer speed if it wasigher than 30 device units. Therefore, all measurements were car-ied out at 60 units.Analytical measurements. The concentration of 2B4 was deter-ined based on an extinction coefficient of A 450–490 5 91 mM21 cm21

or the reduced 2B4–CO complex minus its reduced form in theifference spectrum (22). The concentration of b5 was measuredsing an extinction coefficient of A 424–408nm 5 165 mM21 cm21 for theodium dithionite reduced form minus its oxidized form in the dif-erence spectrum. The concentration of purified Fp was determinedrom its absorbtion spectrum based on an extinction coefficient of

456nm 5 21.4 mM21 cm21 (23). Kinetics of NADPH-dependent reduc-ion of b5 and 2B4 in the presence of benzphetamine was measurednder anaerobic conditions at A 424–408nm and A 450–490nm, respectively.he incubation mixture contained 500 mM KP buffer, pH 7.4, 0.25 gmulgen/liter, 0.5 mM 2B4 monomers, 0.5 mM Fp monomers, and 2M benzphetamine in the case of 2B4 reduction (24) and 0.5 mM Fponomers and 0.5 mM b5 monomers in the case of b5 reduction. The

naerobic system included 50 mM D-glucose, 90 U glucose oxidase/

l, and 2500 U catalase/ ml. The samples were bubbled with CO formin. The reaction was started by adding 1 mM NADPH. The

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89OPTICAL BIOSENSOR STUDY OF KINETIC PARAMETERS FOR COMPLEX FORMATION

inetic curves were fitted using the SpectraLab computer programased on the nonlinear regression method (25). In the presence of b5,he second-electron transfer rate constant was calculated from theifference of the rate constants for NADPH oxidation and superoxidenion generation during one-electron reduction of O2 on 2B4.ADPH oxidation rate was registered at 340 nm and 25°C. The

ncubation mixture contained 500 mM K phosphate buffer, 0.25 gmulgen/liter, pH 7.4, 0.5 mM 2B4 monomers, 0.5 mM Fp monomers,nd 0.5 mM b5 monomers. The reaction was initiated by addition of.3 mM NADPH. The extinction coefficient for NADPH was 6.22M21 cm21. Superoxide anion generation rate was measured by

egistering the superoxide dismutase (SOD)-sensitive reduction ratef 50 mM succinilated cytochrome c as described by Zhukov andrchakov (26). The reaction was started by addition of 0.3 mMADPH. Registration of optical density was carried out at 550 nm

or 1 min, and then 140 U SOD/ml was added. The molar extinctionoefficient of reduced cytochrome c at 550 nm was 21 mM21 cm21

T 5 25°C).

ESULTS AND DISCUSSION

In contrast to 2B4 and Fp, which were monomerizedn low-ionic-strength KP buffers (16), the monomeriza-ion of b5 could only be accomplished with 500 mM KPuffer (27). Since the data on buffer ionic strengthnfluence on 2B4/Fp are controversial (9, 28–30), ex-eriments were conducted to study the influence ofarying KP concentrations on catalytic activity of MRSnd the stimulatory effect of b5 within MRS. The data

TAB

Influence of d- or t-b5 on Benzph

MRS/KP buffer

d-2B4 1 d-Fp (k, s21)

100 mM 500

-b5 0.11 6 0.02 0.09 61d-b5 0.16 6 0.01 0.14 61t-b5 0.11 6 0.01 0.10 6

Note. Incubation mixture contained 100 or 500 KP buffer, pHenzphetamine, 2 mM NADPH. T 5 25°C.

TAB

Relation between Complex Formation and Elec

Pairs k on 3 106 M21 s21 k off (s21)

-2B4 1 d-Fp 0.45 6 0.20 0.20 6 0.10-2B4im 1 t-Fp 0-Fpim 1 d-2B4 0.50 6 0.20 0.30 6 0.10-2B4im 1 d-Fp 0-Fpim 1 t-2B4 4.0 6 2.0 0.30 6 0.10-2B4im 1 t-Fp 0-Fpim 1 t-2B4 1.5 6 0.4 0.40 6 0.10-2B4 1 d-2B4 0.06 6 0.04 0.13 6 0.07-2B4 1 t-2B4 0.3 6 0.1 0.3 6 0.1-2B4 1 t-2B4 1.8 6 0.2 0.20 6 0.05

(t)-Fp 1 d(t)-Fp 0

re presented in Table I. It can be seen that the benz-hetamine hydroxylation rate is unaffected by KP buff-rs of various ionic strength up to 500 mM. In view ofRS activity independence of buffer ionic strength KP,

he complex formation kinetic constants for 2B4, Fp,nd b5 were measured in 500 mM KP buffer, pH 7.4,hroughout.The binding curves for the redox partnerstudied (Figs. 1–4) are very similar: There is an in-rease in the association signal after the addition of theigand (d-Fp or d-2B4) and a decrease of the signalfter buffer addition—evidence for the occurrence ofissociation. To clarify the role of positively chargedroups of proteins in complex formation, either of therotein partners was alternatively immobilized,hrough its amino groups, on the dextran layer. If theinetic constants upon alternative immobilization ofedox partners were essentially the same, they wereresented as one line in Tables II and III; if theyiffered, both values were given—for either partner,eparately.2B4–Fp complex formation. The binding curves for

he full-length and truncated forms of 2B4/Fp pairs arehown in Figs. 1 and 2 and the k on and k off constants inable II. Irrespective of whether the proteins were

ull-length or truncated, their k on values were in the

I

mine N-Demethylation Rate (k)

t-2B4 1 d-Fp (k, s21)

100 mM 500 mM

02 0.027 6 0.002 0.024 6 0.00101 0.034 6 0.003 0.032 6 0.00102 0.025 6 0.002 0.023 6 0.001

4, with 0.25 g Emulgen 913/liter, 1 mM of each protein, 2 mM

II

n Transfer Constants for 2B4/Fp Redox Pairs

K eq 3 106 M21 k e (s21)Productive

complexes (%)

2.2 6 0.98 0.47 6 0.25 100

1.67 6 0.80 0.003 6 0.001 1

12.5 6 7.8 0.020 6 0.003 6

3.7 6 1.3 0.003 6 0.001 0.80.45 6 0.381.0 6 0.59.1 6 2.5

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90 IVANOV ET AL.

rder of (0.3–4.0) 3 106 M21 s21, indicating the complexormation rate to be diffusive-limited (31). The dissoci-tion rate constant for the 2B4/Fp pair was found to be.1–0.4 s21.The binding curves obtained for the full-length

airs—d-2B4im/d-Fp and d-Fpim/d-2B4—are presentedn Figs. 1a and 2a, respectively. Table II presents the

on and k off values calculated from the redox partners’inding curves. The k on and k off values for the d-2B4im/-Fp and d-Fpim/d-2B4 pairs were similar. The fact thatinetic constants for d-2B4/d-Fp did not depend on theartner immobilized is interpreted as evidence for the

TAB

Relation between Complex Formation and Elec

Pairs k on 3 106 M21 s21 k off (s21)

-2B4 1 d-b5 1.2 6 0.5 0.4 6 0.2-2B4 1 t-b5 0-2B4im 1 d-b5 0-b5im 1 t-2B4 4.3 6 1.3 0.5 6 0.1-2B4im 1 t-b5 0-b5im 1 t-2B4 2.8 6 1.2 0.5 6 0.1(t)-b5 1 d(t)-b5 0

IG. 1. The binding of d-Fp or t-Fp to the immobilized d-2B4 (a)nd to immobilized t-2B4 (b). The incubation mixture contained 500M KP buffer (pH 7.4) with 0.25 g Emulgen 913/liter, T 5 25°C.rrows 1 and 2 indicate addition of d- or t-Fp (0.5 mM) and buffer,

cespectively. Solid line, full-length protein; broken line, truncatedrotein.

bsence of electrostatic interactions upon complex for-ation between d-2B4 and d-Fp in 500 mM KP buffer,

H 7.4; moreover, the dominant role of hydrophobicnteractions for this pair is apparent—in accord withhe literature data (9). It was of interest to compare theesults of our biosensor studies with those reported inhe literature. The kinetic data for the d-2B4/d-Fp pro-ein pair, as obtained with Hepes buffer by the fluores-ence quench assay, had been reported earlier (k on 5.5 3 105 M21 s21 and k off 5 0.025 s21) (5). One can seehat our k on for the d-Fpim/d-2B4 pair compares favor-bly with the authors’ k on value. The discrepancy, byne order of magnitude, between the k off constants isossibly due to the use of a higher-ionic-strength buffern this study.

To elucidate the role of membraneous fragments ofB4 in productive complex formation of this proteinith its redox partners, a comparative study of t- and-2B4 with their full-length and truncated redox part-ers was undertaken (Table II). Experiments with im-obilized t-2B4 failed to register its complexing with

-Fp (Fig. 1b), while in the course of d-2B4 interactionith its full-length protein partner the complex forma-

ion did occur. On the other hand, immobilized d-Fpas able to interact with t-2B4 (Fig. 2a). This means

hat unlike with the t-2B4/d-Fp pair, the chargedroups of t-2B4 are absolutely necessary for its com-lexing with d-Fp. The same situation was observedith the d-2B4/t-Fp and t-2B4/t-Fp complexes. Com-lex formation occurred regardless of whether d- or-Fp was immobilized. Thus the positively chargedroups of t-2B4 are absolutely necessary for its com-lex formation with d- or t-Fp.Interestingly, d- and t-2B4 may form complexes be-

ween themselves, with the rate constants for complexormation and decay comparable with those for 2B4/Fpairs. The kinetic constants for complex formation be-ween d- and t-2B4 are represented in Table II. For d-r t-Fp/d- or t-Fp no complex formation was registered.The kinetic data for the full-length and truncated

airs were compared with their electron transfer rate

III

n Transfer Constants for 2B4/b5 Redox Pairs

K eq 3 106 M21 k e (s21)Productive

complexes (%)

3.0 6 2.0 1.00 6 0.03 1000

8.6 6 7.1 0 0

5.6 6 3.1 0 0

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91OPTICAL BIOSENSOR STUDY OF KINETIC PARAMETERS FOR COMPLEX FORMATION

as 0.2 6 0.1 s21 and the electron transfer rate con-tant, k e, was 0.47 6 0.25 s21. Thus, the characteristicife span of the complex (t) is 5.0 6 2.5 s and its electronransfer time is 2.1 6 1.1 s. Comparing these valuesone can see that the life span of the complex is suffi-ient for two electrons to be transferred. The probabil-ty of charge transfer (P 5 t /t e) from d-Fp to d-2B4ithin the life span of the complex is equal to 1 (P

annot exceed 1 by definition); hence, all the complexesormed are productive. If one of the partners is trun-ated, the proportion of productive complexes does notxceed 6%. Thus, the hydrophobic tails of d-2B4 and-Fp are essential for productive complex formation.ther authors also stressed the decisive contribution ofydrophobic interactions between these pairs to thelectron transfer reaction (9).2B4–b5 complex formation. The binding curves for

5/2B4 in 500 mM KP buffer are presented in Figs. 3nd 4. The kinetic constants measured for the full-ength and truncated forms of 2B4/b5 pairs are pre-ented in Table III. Since k on values were in the orderf (1.2–4.3) 3 106 M21 s21, the complex formation rates diffusive-limited (31). The k off value was estimated toe 0.4–0.5 s21.With the full-length d-b5/d-2B4 pair, the complex

ormation and the k on and k off values did not depend onhich of the partners was immobilized. This means

hat for d-2B4/d-b5 (500 mM KP buffer) charge inter-

IG. 2. The binding of d- or t-2B4 to the immobilized d-Fp (a) ando the immobilized t-Fp (b) . The incubation mixture contained 500M K phosphate buffer (pH 7.4) with 0.25 g Emulgen 913/liter, T 5

5°C. Arrows 1 and 2 indicate addition of d- or t-2B4 (0.5 mM) and

de

uffer, respectively. Solid line, full-length protein; broken line, trun-ated protein.

ctions have no role to play in complex formation: Inhis case, the complexing is largely determined by hy-rophobic interactions.To further elucidate the role of membraneous tails of

edox partners, we investigated the binding between-b5im and d-2B4 and, also, between d-2B4im and t-b5.or these pairs, no complex formation was registered

Figs. 3a and 4b). This means that the hydrophobicembraneous tail of b5 plays a dominant role in its

omplexing with d-2B4. The interaction between d- or-b5im with t-2B4 was also studied and in both cases theinding was registered (Fig. 4). One can see that the k on

nd k off values for the complexes of d- or t-b5im with-2B4 are close to the appropriate values for the d-b5im/-2B4 pair (Table III). The binding of the d- or t-b5im/-2B4 pair was found to be dependent on the partnereing immobilized. As seen from Table II and Fig. 3b,o t-2B4im complexes with d- or t-b5 were registered.hus the positively charged groups of t-2B4 are abso-

utely necessary for its interaction with d- or t-b5, asell as with d- or t-Fp, while the interactions between

he t-2B4/d- and t-b5 pairs are due to electrostaticorces. Table II presents the k off values for these pairss well as their electron transfer constants. Comparinghe two constants, one can see that the life span of the

IG. 3. The binding of d-b5 or t-b5 to the immobilized d-2B4 (a) ando immobilized t-2B4 (b). The incubation mixture contained 500 mM

phosphate buffer (pH 7.4) with 0.25 g Emulgen 913/liter, T 55°C. Arrows 1 and 2 indicate addition of d- or t-b5 (0.5 mM) anduffer, respectively. Solid line, full-length protein; broken line, trun-ated protein.

-2B4–d-b5 complex is higher than its interproteinlectron transfer time. Therefore, all the complexes

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92 IVANOV ET AL.

ormed by the full-length enzymes are productive.oreover, within the life span of the complex the trans-

ort of no less than two electrons is realized. If one ofhe interacting proteins was truncated, no productiveomplex formation was observed. Thus, the hydropho-ic tails of d-b5 and d-2B4 play a dominant role inroductive complex formation of these proteins. Inter-stingly, no complexing of d- or t-b5 with one anotheras observed; nor did it occur in the case of d- and t-Fp.Fp–b5 interaction. We have also studied the possi-

ility of complex formation between d- or t-Fp and d- or-b5, but failed to reveal any compexes, irrespective ofhich of the partners was immobilized (the binding

urves are not shown). At the same time, in all possibleombinations of the full-length and truncated pairs,he rate constant for the interprotein electron transferemained unchanged—(0.4 6 0.1) s21. Thus, the elec-ron transfer in d- or t-Fp/d- or t-b5 was effected nothrough complex formation but due to random colli-ion.To elucidate the localization of docking sites of d-2B4

omplexes with d-Fp and d-b5, we have also exploredhe competitive kinetics of complex formation of theull-length d-Fp and d-b5 with immobilized d-2B4. Atrst d-Fp was added to the measuring cuvette up to theaturation level, and then d-b5 was introduced (Fig. 5).he comparison of the binding curve for d-2B4im/d-b5

IG. 4. The binding of d- or t-2B4 to immobilized d-b5 (a) and theinding of d- or t-2B4 to immobilized t-b5 (b). The incubation mixtureontained 500 mM K phosphate buffer (pH 7.4) with 0.25 g Emulgen13/liter, T 5 25°C. Arrows 1 and 2 indicate addition of d- or t-2B40.5 mM) and buffer, respectively. Solid line, full-length protein;roken line, truncated protein.

on the background of the Fp-saturated d-2B4) with theinding curve of d-2B4im/d-b5 in the absence of d-Fp

2t

evealed no distinctions between the two curves. Bynalogous procedure, the d-2B4/d-Fp pair (on the back-round of d-b5-saturated d-2B4im) was compared withhe appropriate pair of proteins but without d-b5 sat-ration of d-2B4im, and again no significant distinc-ions between the binding curves were observed (Fig.). The data provide evidence for the absence of com-etition between d-Fp and d-b5 for the binding site of-2B4 and allow us to conclude that the binding of-2B4 to d-Fp and d-b5 occurs at different sites.Based on our data on the interaction of the full-

ength forms of d-2B4, d-Fp, and d-b5, the electronransfer model for this complex was schematically rep-esented (Fig. 6). Apparently, d-2B4 is able to formompexes with d-Fp and d-b5, whereas with the d-Fp/-b5 pair no compex formation is possible. Thus, withhe reconstituted microsomal system in action, d-b5an acquire one electron—due to its random collisionsith d-Fp—and to pass it over to d-2B4 upon complex

ormation with this latter protein.

ONCLUSION

The real-time optical biosensor study on the interac-ions and complexing between 2B4, Fp, and b5 wasarried out. The kinetic rate constants k on and k off, forhe complexes formed, as well as their affinities, wereetermined. The life spans of the complexes were in therder of a few seconds. The dominant role of the hydro-hobic membranous tails of 2B4, Fp, and b5 in produc-

IG. 5. The binding of d-b5 with immobilized d-2B4 in the presencef the d-Fp (a) and d-Fp with immobilized d-2B4 in the presence of-b5 (b). The incubation mixture contained 500 mM K phosphateuffer (pH 7.4) with 0.25 g Emulgen 913/liter, T 5 25°C. Arrows 1,

, and 3 indicate addition of d-Fp, d-b5, (1.5 mM), and buffer, respec-ively.

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93OPTICAL BIOSENSOR STUDY OF KINETIC PARAMETERS FOR COMPLEX FORMATION

ive complex formation was demonstrated. It wasound that the interprotein electronic transfer mayccur not only through complex formation but also dueo the random collision of protein partners.

CKNOWLEDGMENTS

We thank Dr. J. J. Ramsden for valuable discussions and Drs. G. P.uznetzova and N. F. Samenkova for providing protein prepara-

ions. This work was supported by RFBR Grant N 95-04-12515a, byNTAS grant 961549, and by INCO-COPERNICUS GrantL965070.

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