Effect of the Addition of a Nonaqueous Polar Solvent(Glycerol) on Enzymatic Catalysis in Reverse Micelles.Hydrolysis of 2-Naphthyl Acetate by r-Chymotrypsin

R. Darıo Falcone,† M. Alicia Biasutti,† N. Mariano Correa,† Juana J. Silber,*,†

Eduardo Lissi,‡ and Elsa Abuin‡

Departamento de Quımica, Universidad Nacional de Rıo Cuarto,Agencia Postal N°3 (5800) Rıo Cuarto, Argentina, and Facultad de Quımica y Biologıa,

Universidad de Santiago de Chile, Casilla 40-Correo 33, Santiago, Chile

Received November 30, 2003. In Final Form: April 24, 2004

The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by R-chymotrypsin (R-CT), in reversemicellar solutions formed by glycerol (GY)-water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate(AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of thisreaction with that observed in micelles with water in the core, as well as in the corresponding homogeneousmedia, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in bothhomogeneous media (water and GY-water, 38% v/v mixture). In every media, R-CT was characterized bythe absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of itstryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellarsystems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover,the data obtained allowed the evaluation of the 2-NA partition constant between the organic and themicellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increasein the catalytic properties of R-CT. The fluorescence anisotropy studies in the different media show thatthe addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GYaddition to the reverse micellar aggregates results in a decrease of the conformational mobility of R-CT,which leads to an increase of the enzyme stability and activity.

Introduction

Several biological phenomena occur at interfaces ratherthan in homogeneous solution. In particular, interface/protein interactions play a key role in reactions involvingmembrane-bound proteins. In this sense, even whenreverse micelles constitute an oversimplified model, thevery large interfacial region provided by these systemscan be expected to enhance some effects, such as hydrogenbond interactions between peptide bonds, as a result ofthe fact that in these media the amphipatic essence of abiological membrane is preserved.1

The water dissolved in the inner pool of sodium bis-(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles dif-fers from bulk water in most of its physicochemicalproperties (acidity, microviscosity, polarity, etc.). Forexample, at low W0 ) [H2O]/[AOT] values, the viscosityof the water inside the reverse micelles in aliphatichydrocarbons is nearly 200 times higher than that of bulkwater, and it behaves like that of the biological mem-branes. In this sense, these supramolecular assembliesare useful for membrane mimetic studies.2-7 Moreover,these micelles offer the possibility of carrying out enzy-

matic reactions in nonaqueous media, offering newpossibilities for producing useful chemicals.8

Many studies of enzyme kinetics in reverse micellarsolutions have been reported.3,4,9-19 In most of them,R-chymotrypsin (R-CT),9,15,17,18,20-25 a hydrophilic andglobular enzyme that is totally associated to the micellesand substrates partitioned between the micelles and theexternal solvent, was used. In these cases, it was foundthat the enzymatic activity is often much higher than theone obtained in an aqueous buffer solution.

On the other hand, the reactivity of R-CT was studiedin different reverse micelles systems, using substratesthat are completely incorporated in the water pool.26,27

* Corresponding author. E-mail: jsilber@exa.unrc.edu.ar.† Universidad Nacional de Rıo Cuarto.‡ Universidad de Santiago de Chile.(1) Davis, D. M.; Mcloskey, D.; Birch, D. J. S.; Gellert, P. R.; Kittlety,

R. S.; Swart, R. M. Biophys. Chem. 1996, 60, 63.(2) (a) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. Adv. Colloid

Interface Sci. 1999, 82, 189. (b) Biasutti, M. A.; Correa, N. M.; Silber,J. J. Curr. Top. Colloid Interface Sci. 1999, 3, 35.

(3) De, T. K.; Maitra, A. Adv. Colloid Interface Sci. 1995, 59, 95.(4) Moulik, S. P.; Paul, B. K. Adv. Colloid Interface Sci. 1998, 78, 99.(5) Martinek, K.; Levashov, A. V.; Klyachko, N.; Khmelnitski, Y. L.;

Berezin, I. Eur. J. Biochem. 1986, 155, 453.(6) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim.

Biophys. Acta 1988, 947, 209.(7) Carvalho, C. M. L.; Aires-Barros, M. R.; Cabral, J. M. S. Langmuir

2000, 16, 3082.

(8) Krishna, S. H. Biotechnol. Adv. 2002, 20, 239.(9) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 6731.(10) Verhaert, R. M. D.; Hilhorst, R.; Vernue, M.; Schaafsma, T. J.;

Veeger, C. Eur. J. Biochem. 1990, 187, 59.(11) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084.(12) Aguilar, L. F.; Abuin, E.; Lissi, E. Arch. Biochem. Biophys. 2001,

388, 231.(13) (a) Sarcar, S.; Munshi, N.; Jain, T. K.; Maitra, A. Biotechnol.

Bioeng. 1992, 39, 474. (b) Sarcar, S.; Munshi, N.; Jain, T. K.; Maitra,A. Colloids Surf., A 1994, 88, 169.

(14) Das, S.; Mozumdar, S.; Maitra, A. J. Colloid Interface Sci. 2000,230, 328.

(15) Miyake, Y. Colloids Surf., A 1996, 109, 255.(16) Setti, L.; Fevereiro, P.; Melo, E. P.; Pifferri, P. G.; Cabral, J. M.

S.; Aires-Barros, M. R. Appl. Biochem. Biotechnol. 1995, 55, 207.(17) Ruckenstein, E.; Karpe, P. J. Colloid Interface Sci. 1990, 139,

408.(18) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239.(19) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063.(20) Miyake, Y.; Owari, T.; Ishiga, F.; Teramoto, M. J. Chem. Soc.,

Faraday Trans. 1994, 90, 979.(21) Rarly, R. V.; Bec, N.; Klyachko, N. L.; Levashov, A. V.; Balny,

C. Biotechnol. Bioeng. 1998, 57, 552.(22) Andrade, S. M.; Costa, S. M. E. J. Mol. Struct. 2001, 565, 219.(23) Serralheiro, M. L. M.; Cabral, J. M. S. J. Mol. Catal. B: Enzym.

1999, 7, 191.(24) Castro, G. R. Enzyme Microb. Technol. 2000, 27, 143.(25) Martinek, K.; Levashov, A. V.; Klyashko, N. L.; Pantin, V. I.;

Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277.

5732 Langmuir 2004, 20, 5732-5737

10.1021/la036243x CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 06/05/2004

For these cases, it was found that the reactivity (kcat) ofR-CT is very similar in the reverse micelle systems thanin bulk water. However, the Michaelis constant (KM)increases significantly in the microemulsions, and thiseffectively compensates for the increase in the reactionrate due to the concentrative effect of the water-solublesubstratesand R-CT inthewaterpool of theaggregates.26,27

The majority of the studies performed on the catalyticbehavior of R-CT in reverse micellar media have beendone using water as the polar solvent encapsulated in thecore of the aggregates. However, it is known that thesesystems can dissolve other solvents such as glycerol (GY),ethyleneglycol, and formamide as well as their binarymixtures with water.28-31 In particular, as a result of thestabilizing effect that bulk GY has on proteins32 it isinteresting to study the effect that this polar solvent hason enzyme encapsulated inside the polar core of the reversemicelle systems. In a recent work,21 it has been shownthat, when a mixture of GY and water is added to a AOT/isooctane reverse micellar solution, the thermostabilityof R-CT increases as compared with the one found in water/AOT/isooctane. It was concluded that, when GY is addedas a water-miscible cosolvent, the micellar matrix struc-ture order increases, decreasing the mobility of the enzymemolecule and, hence, increasing its stability.

Studies by noninvasive techniques such as Fouriertransform infrared spectroscopy and 1H NMR have beenemployed to investigate the solubilization of GY and theGY-water mixture by AOT reverse micellar systems. Itwas found that GY and water interact similarly with AOT,namely, by solvating the surfactant head ions, which isthe polar solvent GY that interacts strongly with AOT.31

Similar results were found using an optical probe.29 Inthe GY-water mixture, the interaction with the surfactantby the mixture is dominated by water.31

The aim of the present work is to determine the influenceof the addition of GY on the hydrolysis of 2-naphthylacetate (2-NA) catalyzed by R-CT to water/AOT/n-heptanereverse micelles and to compare the efficiency of thisreaction with that observed in micellar systems, as wellas in the corresponding homogeneous media. Hence, thehydrolysis rates of 2-NA catalyzed by this enzyme, in bothhomogeneous [water and a GY-water (38% v/v) mixture]and reverse micellar systems [water/AOT/n-heptane anda GY-water (38% v/v) mixture/AOT/n-heptane], have beendetermined by spectroscopic measurements. In addition,the method used allowed for the joint evaluation of thepartitioning of the substrate, KP, between the organic andthe micellar pseudophase and the kinetic parameters: thecatalytic rate constant kcat and the Michaelis constant KMof the enzymatic reaction for R-CT totally incorporated inthe reverse micelles. The enzyme was characterized inthese media through the absorption and emission proper-ties of its tryptophan residues. The effect of the AOTconcentration on the kinetic parameters was determined,at constant W0 ) [H2O]/[AOT] or WM ) [GY + H2O]/[AOT].It is shown that the addition of GY to the micelle interiorresults in an increase in the catalytic properties of R-CT.

Experimental SectionGeneral Information. Absorption spectra were measured

by using a HP 8452A spectrophotometer with a cell holderthermostated at 25.0 ( 0.5 °C. A Hitachi F-2500 apparatus wasemployed for the fluorescence measurements. Fluorescence decaytimes were measured by the time-correlated single-photon-counting technique using Edinburgh Instrument FL-9000 equip-ment. Fluctuations in the pulse and intensity were corrected bymaking an alternate collection of scattering and sample emis-sions. The quality of the fits was determined by the reduced ø2

values.33 The path length of the cell used in absorption andemission experiments was 1 cm.

Stationary anisotropy measurements were performed in a SLMAminco 4800 C spectrofluorometer. In the micellar media,background fluorescence, as well as light scattering, was removedby subtraction of a spectrum recorded on a blank solution.Fluorescence anisotropy ⟨r⟩ values were obtained at 25 °C usingthe expression ⟨r⟩ ) IVV - GIVH/(IVV + 2GIVH), where IVV and IVHare the vertically and horizontally polarized components of R-CTtryptophan residue emissions after excitation by vertically polar-ized light and G is the sensitivity factor of the detection system.34

Materials. AOT, from Sigma (99%), was dried under a reducedpressure and kept under a vacuum over P2O5 until its use. Theabsence of acidic impurities was carefully checked using 1-methyl-8-oxyquinolinium betaine as the indicator,29,35,36 because thoseimpurities can significantly affect the pH of the dispersed aqueousphase.26

R-CT, molecular weight 24 800, from bovine pancreas (Sigma),and 2-NA (Sigma) were used as received. n-Heptane (SintorganHPLC) and GY from Merck (fluorescence spectroscopy quality)were used without purification. Ultrapure water was obtainedfrom a Labonco equipment model 90901-01. The GY-watermixture used was 38% (v/v). The pH of the bulk solutions (wateror GY mixture) has been maintained at 8.7 by using a 20 mMphosphate buffer. In the reverse micelles media, it is known thatthe pH cannot be measured inside the polar core of theaggregate.37,38 A meaningful approximation to the pH within theaqueous pseudophase of the reverse micelles can be made usinga pure source of AOT and to have sufficient buffering capacityin the bulk solutions (i.e., such that the observed rates areindependent of the buffer concentrations).26 In this sense, thevalue of the pH inside the polar core is referenced to thehomogeneous buffer solution, and it is called pHext.

Procedures and Kinetics. The solutions of AOT in n-heptanewerepreparedbyweighinganddilution.Themolarratiosbetweenwater or GY-water and AOT are defined as W0 ) [H2O]/[AOT]and WM ) [GY + H2O]/[AOT], respectively. The addition of thepolar solvent (water or GY-water) to the micellar system wasperformed using a calibrated microsyringe.

Partition constants for 2-NA between n-heptane and the polarmedium, water or the GY-water mixture, considered weredetermined by the hand-shaking method, using UV-visiblespectroscopy to record the decrease of the absorbance of 2-NA inn-heptane after mixing with the polar solvent considered at 25.0( 0.5 °C.

Reactions were followed spectrophotometrically by the increaseat the maximum of the product (2-naphthol, 2-N) absorptionband (λmax ) 327 nm) at 25.0 ( 0.5 °C. To start a kinetic run inhomogeneousmedia,astock solutionof2-NAinthebuffer solution(water or the GY-water mixture) was added into a thermostatedcell containing the R-CT in the same buffer. The concentrationsof 2-NA and R-CT in the reaction media were around 10-4 M and10-6 M, respectively. In the micellar media, the stoppered cellwas filled with a fixed volume of AOT in n-heptane with thesubstrate. WM or W0 ) 13.2 was reached by adding the buffer(26) Fletcher, P. D. I.; Freedman, R. B.; Mead, J.; Oldfield, C.;

Robinson, B. H. Colloids Surf. 1984, 10, 193.(27) Fletcher, P. D. I.; Rees, G. D.; Robinson, B. H.; Freedman, R. B.

Biochim. Biophys. Acta 1985, 832, 204.(28) Avyard, R.; Binks, B. P.; Fletcher, P. D. I.; Kirk, A. J.; Swansbury,

P. Langmuir 1993, 9, 523.(29) Falcone, R. D.; Correa, N. M.; Biasutti, M. A.; Silber, J. J.

Langmuir 2000, 16, 3070.(30) Novaki, L. P.; Correa, N. M.; Silber, J. J.; El Seoud, O. A.

Langmuir 2000, 16, 5573.(31) El Seoud, O. A.; Correa, N. M.; Novaki, L. P. Langmuir 2001,

17, 1847.(32) Gekko, K.; Timashef, S. N. Biochemistry 1981, 20, 4667.

(33) O’Connor, D. V.; Phillips, D. Time-Correlated Single PhotonCounting; Academic Press: New York, 1983; Chapter 6.

(34) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;Kluwer Academic: New York, 1999; Chapter 16, Protein fluorescence.

(35) Correa, N. M.; Biasutti, M. A.; Silber, J. J. J. Colloid InterfaceSci. 1995, 172, 71.

(36) Correa, N. M.; Biasutti, M. A.; Silber, J. J. J. Colloid InterfaceSci. 1996, 184, 570.

(37) Smith, R.; Luisi, P. L. Helv. Chim. Acta 1980, 63, 2302.(38) Falcone, R. D.; Correa, N. M.; Biasutti, M. A.; Silber, J. J.

Langmuir 2002, 18, 2039.

Enzymatic Catalysis in Reverse Micelles Langmuir, Vol. 20, No. 14, 2004 5733

solution. After thermostating the cell, the enzymatic reactionwas initiated by the addition of a volume of R-CT dissolved inthe reverse micelles to have 3 mL of micellar solution with thedesired 2-NA and R-CT concentrations.9

The hydrolysis of 2-NA catalyzed by R-CT, which in the reversemicellar system is totally incorporated to the micellar pseudo-phase, follows the Michaelis-Menten mechanism10,11 depictedin eq 1:

Applying the steady-state approximation to E-S, the rate lawgiven in eq 2 is obtained:

where v0 is the initial reaction rate (in M s-1 units), [E] is theanalytical enzyme concentration, [S] is the analytical concentra-tion of the substrate, kcat is the catalytic rate constant, and KMis the Michaelis constant defined by eq 3:

Equation 2 can be rearranged into a form that is amenable todata analysis by linear regression, which is known as theLineweaver-Burk equation (eq 4):

Equation 4 directly provides kcat, from the reciprocal of theintercept, and the catalytic efficiency kcat/kM, from the reciprocalof the slope. KM is obtained from the slope/intercept ratio.

The absorbance was recorded as a function of time, and theinitial velocity (v0) was obtained from the slope of [2-N] versustime profiles. The formation of 2-NA was linearly dependent onthe reaction time during the first 20 min of reaction. The v0values were plotted according to the Lineweaver-Burk relation-ship (eq 4).

The pooled standard deviation of the kinetic data, usingdifferent samples, was less than 5%.

ResultsThe hydrolysis of 2-NA catalyzed by R-CT in homoge-

neous media, either water or the GY-water mixture, andin reverse micelles of water/AOT/n-heptane or GY-water/AOT/n-heptane produces 2-N (eq 5) in quantitative yieldsas shown by UV-visible spectroscopic analysis of thereaction mixture.

The absorption spectra taken at different times ofreaction show a clear isosbestic point, evidencing the lackof intermediates and product decomposition. The hy-drolysis of 2-NA in the absence of the enzyme wasevaluated in every reaction medium, yielding half reactiontimes (t1/2) of around 420 min in buffer and 90 min in theGY-buffer mixture. In the reverse micellar systems, no

product was detected for at least 4 h. On the other hand,when the reaction was carried out in the presence of R-CT(see the following), the values of t1/2 were around 0.5 minand, therefore, it can be concluded that there is no influenceof the nonenzymatic reaction to the measured kineticparameters.

Preliminary studies (results not shown) varying severalexperimental conditions such as the GY-water composi-tion, pHext, and WM demonstrated that the maximumcatalytic efficiency of the enzyme is obtained, in bothhomogeneous and microheterogeneous media, using GY-water (38% v/v), pHext ∼ 8.7, and WM ) 13.5. Therefore,these conditions were used for all subsequent measure-ments reported in the paper.

Prior to studying the enzymatic reaction in the GY-water mixture and GY-water/AOT/n-heptane solutions,the stability of R-CT was tested by absorption and emissionspectroscopy, and the results are shown in Table 1. Inthis table are also gathered the values of the fluorescencelifetimes and the steady-state anisotropy data.

Reaction in Homogeneous Phases (Water or GY-Water). Figure 1 shows typical results obtained bytreating the data according to the Lineweaver-Burk plot(eq 4). The linearity of the plot indicates that, under thecondition employed, the Michaelis-Menten mechanismapplies in the GY-water mixture, as it has been previouslyshown for the reaction in water at pH 8.9.20 From theslope and the intercept of the line in Figure 1, values ofthe experimental kinetics parameters, kcat

exp and (KMexp)bulk,

were calculated. The values obtained are gathered in Table2. Also, the results obtained in water (pH ) 8.7) areincluded for comparison. As can be observed (Table 2),the catalytic efficiency of the enzyme, given by the ratioof (kcat

exp/KMexp)bulk, is higher in the GY-water mixture than

in water. The increase of nearly a factor of eight is dueto both an increase in kcat

exp and a reduction in (KMexp)bulk.

ReactioninWater/AOT/n-HeptaneandGY-Water/AOT/n-Heptane Microemulsions. Although this en-

Table 1. Absorption and Emission Maxima, Fluorescence Lifetimes (λexc ) 295 nm, λem ) 334 nm) and Steady-StateAnisotropy ⟨r⟩ (λexc ) 295 nm) of r-CT in Different Mediaa

medium λabsmax (nm) λem

max (nm) τ1 (ns) τ2 (ns) τ3 (ns) ⟨r⟩

water 282 333 0.72 ( 0.04 2.7 ( 0.2 5.7 ( 0.6 0.108 ( 0.005GY-water (38% v/v) 282 334 0.94 ( 0.06 3.1 ( 0.3 6.2 ( 0.8 0.120 ( 0.006water/AOT/n-heptane 282 334 0.17 ( 0.03 1.8 ( 0.1 4.9 ( 0.1 0.113 ( 0.006GY-water (38% v/v)/AOT/n-heptane 282 334 0.16 ( 0.02. 1.7 ( 0.1 5.2 ( 0.2 0.161 ( 0.008a [R-CT] ) 1 × 10-5 M, [AOT] ) 0.4 M, WM ) 13.2; pHext ) 8.7.

Figure 1. Lineweaver-Burk plot for the R-CT catalyzedhydrolysis of 2-NA in GY-water (38% v/v), pH ) 8.7, [R-CT]) 2.5 × 10-6 M.

E + S y\zk1

k-1E-S 98

kcatE + P (1)

v0 )kcat[E][S]

KM[S](2)

KM ) (k-1 + kcat)/k1 (3)

[E]/v0 ) (1/kcat) + (KM/kcat)/[S] (4)

5734 Langmuir, Vol. 20, No. 14, 2004 Falcone et al.

zymatic reaction has been already studied by otherauthors18 in water/AOT/n-heptane at pH ) 8.9, in thepresent work the hydrolysis was also studied in the samesystem at pHext ∼ 8.7 to compare with the results obtainedin GY-water/AOT/n-heptane microemulsions under thesame experimental conditions, such as ionic strength,enzyme source, buffer used, and so forth. Representativeresults obtained for the effect of the AOT concentrationon the relationship between v0/[R-CT] and the analyticalconcentration of 2-NA ([2-NA]T) at W0 or M ) 13.2 are shownin Figure 2A,B for water/AOT/n-heptane and GY-water/AOT/n-heptane microemulsions, respectively. As can beseen from Figure 2, the v0/[R-CT] ratio is lower when wateris used as the polar solvent. Similarly, enzyme satu-ration with the substrate is reached at lower [2-NA]T

values, indicating a tendency to lower values of (KMexp)mic

(Michaelis constant determined in terms of the analyticalconcentration of the substrate) for these reverse micelles.Also, for both systems it can be seen that an increase inthe surfactant concentration produces a decrease in v0/

[R-CT] over the whole range of concentrations studied.Moreover, in the GY-water/AOT/n-heptane system thisratio reaches similar values at [AOT] > 0.25 M, while inthe water/AOT/n-heptane system, v0/[R-CT] clearly di-minishes over the entire [AOT] range considered.

The data shown in Figure 2 were plotted according toeq 4. A representative example of the plots obtained forGY-water/AOT/n-heptane is shown in Figure 3. Thelinearity of the plots at every [AOT] considered indicatesthat the classical Michaelis-Menten mechanism is validfor R-CT in the GY-water/AOT/n-heptane microemulsion,as it has been observed in water/AOT/n-heptane, for thisand other related reactions.9,12 From Figure 3, values ofthe experimental kinetic parameters in the microhetero-geneous media at different surfactant concentrations canbe obtained, and the results are shown in Table 3.

To assess the activity of the substrate in the differenthomogeneous solvents employed (n-heptane, water, andGY-water), we have determined its partition constantbetween n-heptane and the polar solvents. Values ofKwater/hep

bulk and the corresponding for the GY-water mix-ture, KGY-water/hep

bulk , were 3.6 × 10-3 and 6.6 × 10-3,respectively. These values indicate that, at a givenconcentration, the activity of 2-NA is 280 times higher inwater than in n-heptane and that the presence of GYreduces this value by a factor of 1.8.

DiscussionFrom the data given in Table 1, it can be concluded that

the absorption and emission maxima of the protein arevery similar in homogeneous and micellar media. On theother hand, when the spectrum of the enzyme was recordedin a water solution of 8 M urea (results not shown), wherethe quaternary and tertiary structures are broken andthe tryptophan residues are fully exposed to water,34 thefluorescence maxima is red-shifted to 350 nm matchingthe value of the emission maxima of free tryptophan inwater or the GY-water mixture. Therefore, it can be

Table 2. Summary of Experimental Kinetic Parameters of the Enzymatic Reaction in Homogeneous Media

mediumkcat

exp

(s-1) × 102(KM

exp)bulk

(M) × 103(kcat

exp/KMexp)bulk

(M-1 s-1)((KM)corr)bulk

(M) × 103[kcat

exp/(KM)corr]bulk

(s-1 M-1)

water 2.10 ( 0.21 0.57 ( 0.06 36 0.16a 0.1GY-water (38% v/v) 8.80 ( 0.88 0.31 ( 0.03 284 0.05a 1.8

a Correction according to eq 12.

Figure 2. Effect of AOT concentration on the relationshipbetween the initial rate of reaction (per enzyme) and theanalytical concentration of 2-NA in reverse micelles of (A)n-heptane/AOT/water at W0 ) 13.2 with [AOT] of (0) 0.10, (9)0.25, and (b) 0.40 M and [R-CT] ) 3 × 10-6 M and (B) GY-water (38% v/v)/AOT/n-heptane at WM ) 13.2 with [AOT] of (0)0.10, (O) 0.20, (9) 0.25, and (b) 0.40 M and [R-CT] ) 2.5 × 10-6

M.

Figure 3. Effect of AOT concentration on the Lineweaver-Burk plot for the R-CT-catalyzed hydrolysis of 2-NA in reversemicellar solutions of GY-water (38% v/v)/AOT/n-heptane atWM ) 13.2. [AOT]: (0) 0.10, (O) 0.20, (9) 0.25, and (b) 0.40 Mand [R-CT] ) 2.5 × 10-6 M. pHext ) 8.7.

Enzymatic Catalysis in Reverse Micelles Langmuir, Vol. 20, No. 14, 2004 5735

assumed that the localization of R-CT is the water poolof the reverse micelles, as it was previously found,26,39

and that this encapsulation does not significantly alterthe tertiary structure of R-CT.

With regard to fluorescence lifetimes, the data of Table1 show that, in all the media considered, there are threecomponents, which have been attributed to differentenvironments where the tryptophan residues in theprotein are located: (i) the protein’s surface, (ii) theprotein’s surface but with the indolic rings facing theprotein’s interior, and (iii) in a protein’s more hydrophobicenvironment.22 It must be pointed out that the fluorescencelifetimes of R-CT in the reverse micelles are shorter thanin water or the GY-water mixture. It is known that theemission of tryptophan alone can be self-quenched by anintramolecular process involving the indol ring and theammonium group. Also, the tryptophan residue in R-CTshows evidence of quenching effects promoted by peptideor disulfide bonds.22 In the reverse micellar media, it seemsthat those quenching effects are enhanced probablybecause of the restricted environment where the proteinis forced to reside, with the corresponding diminution ofthe fluorescence mean lifetime.34

As can be seen in Table 1, the value of ⟨r⟩ increases inthe presence of GY, as compared with water in bothhomogeneous and microheterogeneous media. Proteinfluorescence anisotropy is generally recognized as aqualitative measure of macromolecular mobility, and thosefactors, which increase the degree of polarization, are alsoassumed to decrease the mobility of the protein.18 Thedata suggest that the presence of GY, particularly in thereverse micelle media, reduces the mobility of the enzymefluorophores.

As can be observed from Table 2, the efficiency of theenzymatic reaction in homogeneous media is nearly 8times greater in the GY-water mixture than in water,probably as a result of the high viscosity around theenzyme in the organic mixture and, consequently, thelower mobility of the protein (Table 1). Determination ofthe activation parameters could support this proposal.However, these measurements cannot be performed inthe microheterogeneous media, particularly in the pres-ence of polar solvents other than water, because theirstructure and properties are highly dependent on thetemperature.40 Thus, there is not a wide enough temper-ature range to measure these parameters.

The dependence with the AOT concentration found inFigure 2A,B could be due to (i) progressive inactivationof the enzyme by the surfactant, leading to lower kcat valueas [AOT] increases, and (ii) 2-NA partitioning (due to its

greater solubility in n-heptane), leading to a lower localsubstrate concentration within the zone in which thereaction takes place, that is, the micellar pseudophase.This amounts to a simple dilution effect (i.e., to a localconcentration inversely proportional to the surfactantconcentration) when the substrate is totally associated tothe dispersed pseudophase.26,27

From Figure 3, it can be seen that a unique interceptis obtained irrespectively of the surfactant concentration.This fact argues against possibility i as expected for thecase in which the enzyme is totally incorporated to thereverse micelles. This suggests that the differences in thekinetic profiles shown in Figures 2 and 3 are due to thepartitioning of the substrate between the micellarpseudophase and the external solvent (KP). The moremarked dependence with AOT concentration in thepresence of water (Figure 2A,B) allows the inference thatthe value of KP has to be higher in GY-water/AOT/n-heptane than in water/AOT/n-heptane. Furthermore,incorporation of 2-NA to the micellar pseudophase explainsthe increase in (KM

exp)mic observed at high surfactantconcentrations (Table 3).

The partitioning of a solute between the micelles andthe organic solvent can be treated within the frameworkof the pseudophases model. According to the two-pseudophase model, which consider two phases present,the surrounding nonpolar solvent and the microaggre-gates,2 the partitioning of 2-NA between the micelles andthe external solvent pseudophase can be defined as in eq6

with KP as shown in eq 7

With both concentrations, free (f) and bound (b) are givenin terms of local concentrations. If [2-NA]b is the analytical(bulk) concentration of the micelle-bound substrate, eq 8holds

and, hence, KP can be expressed as in eq 9

where [2-NA]b is the analytical concentration of the

(39) Levashov, A. V.; Khmelnitski, Y. L.; Klyashko, N. L.; Chernyak,V. Y.; Martinek, K. Anal. Biochem. 1981, 118, 42.

(40) Ray, S.; Moulik, S. P. Langmuir 1994, 10, 2511.

Table 3. Summary of Experimental Kinetic Parameters of the Enzymatic Reaction in Microheterogeneous Media

[AOT](M)

kcatexp

(s-1) × 102(KM

exp)mic

(M) × 103(kcat

exp/KMexp)mic

(M-1 s-1)((KM)corr)mic

(M) × 103[kcat

exp/(KM)corr]mic

(s-1 M-1)

Water/AOT/n-Heptane Medium0.10 0.24 ( 0.02 2.30 ( 0.23 1.1 2.1a

0.25 0.21 ( 0.02 2.40 ( 0.24 0.8 1.8a

0.40 0.21 ( 0.02 3.50 ( 0.35 0.6 2.3a

average 0.22 ( 0.02 2.1a 1.1

GY-Water/AOT/n-Heptane Medium0.10 6.6 ( 0.3 16 ( 0.8 4.2 12a

0.25 8.2 ( 0.4 31 ( 1.5 2.6 18a

0.40 7.7 ( 0.4 32 ( 1.6 2.4 14a

average 7.5 ( 0.4 15a 5.0a Correction according to eq 11.

2NAf h 2-NAb′ (6)

KP )[2-NA′]b

[2-NA]f(7)

[2-NA′]b )[2-NA]b

[AOT](8)

KP )[2-NA]b

[2-NA]f[AOT](9)

5736 Langmuir, Vol. 20, No. 14, 2004 Falcone et al.

substrate incorporated to the micelles, [2-NA]f is theconcentration of the substrate in n-heptane, and [AOT]is the concentration of the surfactant. This equationapplies at a fixed value of W0 or M and when [2-NA]T ,[AOT].

As was previously described,12 it is possible to determineKP from the kinetic data using eq 10:

where [2-NA]T is the total (analytical) substrate concen-tration and n is the average number of substrate moleculesincorporated into reverse micelles per AOT molecule; n )[2-NA]b/[AOT]. The proposed method is based on theassumption that, at a givenW0 or M and [enzyme], the valueof v0/[enzyme] is determined only by the concentration ofthe substrate in the organic solvent. Because equal [2-NA]fimplies equal n, a simple mass balance leads to eq 10.12

For a set of [2-NA]T and [AOT] values corresponding tothe same v0/[E] value, a plot of the left-hand side of eq 10against [AOT] allows the evaluation of n from the slopeand KP from the slope/intercept ratio. The results obtainedwere KP ) 1.2 ( 0.5 M-1 for the water/AOT/n-heptanesystem and KP ) 3.1 ( 0.8 M-1 for the GY-water/AOT/n-heptane system. One possible explanation of the dif-ferences in KP comes out comparing them with the valuesof KGY-water/hep

bulk and Kwater/hepbulk , which show that the inter-

action of 2-NA with the GY-water mixture is higher thanwith water. A correlation of KP with the bulk partitionconstants is expected only if the determining factor of thedistribution is the solvent-solute interactions.41 In thiscase, because the value of WM is high enough (>10) toensure that there are free polar solvents in the inner coreof the reverse micelles, this correlation seems to be possibleand the trend found in KP for the reverse micelles is thesame as in the bulk solvents.

The experimental kinetics parameters presented inTable 3 show that kcat

exp is practically constant at all thesurfactant concentrations used. It can be considered that,when varying [AOT] but at a constant W, the shape andsize of the reverse micelles are maintained.42 In this sense,there is no reason to expect a variation in kcat with [AOT].It is noticeable that, both in bulk solution and in themicelles, incorporation of GY notably increases the valueof kcat

exp. This is particularly so in the reverse micelles,when this rate constant is nearly 35 times larger in thepresence of GY. On the other hand, (KM

exp)mic increaseswith [AOT] probably as a result of the distribution of thesubstrate between the micelles and the external solvent,as it has been previously observed for the lipase-catalyzedhydrolysis of 2-NA in AOT reverse micelles.12 Thus, thevalues of (KM

exp)mic were corrected by the partitioning of2-NA (KP) with eq 11:12

The results are summarized in Table 3, giving valuespractically independent of the AOT concentration. Con-sequently, the decrease observed in (KM

exp)mic with theAOT concentration is not a dilution effect but is due to thesubstrate partitioning.

With regard to the [kcatexp/(KM)corr]mic ratio (Table 3) as

compared with the one obtained in homogeneous media(kcat

exp/KMexp)bulk (Table 2), it can be seen that the presence of

the micelles reduces the values by factors of 33 and 57 inthe water/AOT and GY-water/AOT systems, respectively.However, as it has been described by Lissi and Abuin,11

a meaningful comparison of the kinetic parametersobtained in reverse micellar solutions with those obtainedin bulk solvents to determine the enzyme inactivation orhyperactivity requires therate lawtobeexpressed in termsof a common thermodynamic substrate activity scale andnot in terms of substrate concentrations. The simplestapproach is to compare the rate constants taking, as areference the external solvent of the reverse micelles. Inother words, the catalytic efficiency of the enzymaticreaction obtained in the bulk solvents, (kcat

exp/KMexp)bulk,

must be corrected by the partitioning of the sub-strate between the organic and bulk polar solvents(Kpolar solvent/hep

bulk ). This correction is shown in eq 12:11

The values of [kcatexp/(KM)corr]bulk obtained are gathered in

Table 2. Taking into account this correction, it is observedthat the catalytic efficiencies (Tables 2 and 3) in themicellar media are higher than those obtained in thecorresponding homogeneous ones. This fact can be at-tributed to the highly structured polar solvents in theinner core of the reverse micelles, which produces a moreviscous microenvironment around the enzyme with adecrease in its mobility (Table 1). The correlation betweenhigher activity and higher rigidity agrees also with thetime-stability previously found for this enzyme.18

Comparing the catalytic efficiencies obtained in bothreverse micellar systems, GY-water/AOT/n-heptane andwater/AOT/n-heptane, it can be seen that the value in theformer one is 5 times higher. This fact can also be explainedby considering that the addition of GY in the reversemicellar aggregates results in a decrease of conformationalmobility of the R-CT (see ⟨r⟩ value in Table 1), which leadsto an increase of the enzyme stability and activity. Theincrement of the enzyme stability has been demonstrated21

to occur with polyols, which easily form a net of hydrogenbonds that fix the protein structure through a more rigidmatrix structure. Also, note that the presence of GYnotably increases the efficiency of the R-CT in homoge-neous media, being the ratio kcat

exp/(KM)corr (Tables 2 and3) similar to the one obtained in water/AOT/n-heptanereverse micelles. Moreover, as shown in Table 1, the valuesof ⟨r⟩ are similar for the mixture GY-water and water/AOT/n-heptane reverse micelles. On the other hand, it isclearly demonstrated that the incorporation of the GY-watermixture in theAOT/n-heptanereversemicelles leadsto a novel and powerful reaction medium for this enzymaticreaction.

Acknowledgment. Financial support in Argentinafrom the Consejo Nacional de Investigaciones Cientıficasy Tecnicas (CONICET), Universidad Nacional de RıoCuarto, Fundacion Antorchas, and Agencia Nacional dePromocion Cientıfica y Tecnica is gratefully acknowledged.Also in Chile Dicyt (USACH), Fondecyt (Project No.1010148), and a collaborative project Setcip (Argentina)/Conicyt (Chile) are thanked. Dr. G. Montich from Uni-versidad Nacional de Cordoba is thanked for allowing usto use the steady-state fluorescence anisotropy equipment.

LA036243X

(41) Silber, J. J.; Falcone, R. D.; Correa, N. M.; Biasutti, M. A.; Abuin,E.; Lissi, E.; Campodonico, P. Langmuir 2003, 19, 2067.

(42) Lissi, E. A.; Engel, D. Langmuir 1992, 8, 452.

[2-NA]T ) nKP

+ n[AOT] (10)

((KM)corr)mic )

(KMexp)mic

1 + KP[AOT](11)

((KM)corr)bulk ) (KM

exp)bulk/Kpolar solvents/hepbulk (12)

Enzymatic Catalysis in Reverse Micelles Langmuir, Vol. 20, No. 14, 2004 5737