Colloids and Surfaces B: Biointerfaces 26 (2002) 291 300 www.elsevier.

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The effect of poly(ethylene glycol) on the activity and structure of glucose-6-phosphate dehydrogenase in solution
Sabrina M. Pancera a, Luis H.M. da Silva b, Watson Loh b, Rosangela Itri c, Adalberto Pessoa Jr d, Denise F.S. Petri a,*
a Instituto de Qu mica, Uni6ersidade de Sa o Paulo, PO Box 26077, 05513 -970 Sa o Paulo, SP, Brazil Instituto de Qu mica, Uni6ersidade Estadual de Campinas, PO Box 6154, 13083 -970, Campinas, SP, Brazil c Instituto de F sica, Uni6ersidade de Sa o Paulo, PO Box 66318, 05315 -970, Sa o Paulo, SP, Brazil d Faculdade de Cie ncias Farmace uticas, Uni6ersidade de Sa o Paulo, PO Box 66083, 05315 -970, Sa o Paulo, SP, Brazil b

Received 21 August 2001; received in revised form 7 November 2001; accepted 2 January 2002

Abstract The effect of poly(ethylene glycol), PEG, on the enzymatic activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the oxidation of glucose-6-phosphate (G-6-P), using NADP + as co-enzyme was investigated. The enzymatic activity was determined by means of spectrophotometry in three different media: pure Tris HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. Comparing the enzymatic activity values measured in pure buffer with those in the polymer solutions, an increase in the enzymatic activity of 20% was observed in the presence of PEG400 as well as in PEG4000. Calorimetric studies indicated the absence of preferential interactions between G-6-PDH and PEG400 or PEG4000. Nevertheless, the interaction enthalpy, DHint, between NADP + and PEG400 and PEG4000 amounted to 9.3 and 26.7 kJ/mol, respectively. Small angle X-ray scattering (SAXS) measurements were performed in a higher concentration range. Data analysis performed from SAXS curves by means of the intra-particle distance distribution function p (r ) and Guinier plots yielded for G-6-PDH , and in PEG4000 solutions, Rg of about in pure buffer and PEG400 solutions radius of gyration, Rg, of about 70 A , . The latter has the same dimension as that found in the dimeric crystallographic structure of G-6-PDH, 40 A evidencing that G-6-PDH preserves its dimeric conguration in PEG4000 solution. On the contrary, different aggregates of G-6-PDH are formed in the presence of either buffer or PEG400. These ndings show that the presence of PEG in solution can exert an effect on the enzyme structure and activity. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Glucose-6-phosphate dehydrogenase; Poly(ethylene glycol); b-Nicotinamide adenine dinucleotide phosphate; Enzymatic activity; Calorimetry; Small angle X-ray scattering

1. Introduction Glucose-6-phosphate dehydrogenase (G-6PDH) (EC1.1.1.49) is the rst enzyme in the pentose phosphate pathway. In the presence of

* Corresponding author. Fax: + 55-11-3815-5579. E -mail address: dfsp@quim.iq.usp.br (D.F.S. Petri).

0927-7765/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 0 1 1 - 5

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the coenzyme b-nicotinamide adenine dinucleotide phosphate (NADP + ) and/or NAD+ it catalyses the oxidation of glucose-6-phosphate (G-6-P). The determination of molecular weight and dimension of G-6-PDH from Leuconostoc mesenteroides is well reported in the literature [1 5]. Studies [2,6,7] on the mechanism of catalytic activity show that His-240 acts as a general base abstracting the proton from C1-hydroxyl of G-6-P, while Asp-177 stabilizes the positive charge formed on His-240 in the transition state and the transfer of the C1-hydride to the pyridine ring of NADP + occurs. It was also proposed that the peptide His-178 plays an important role because it binds the phosphate moiety of G-6-P by hydrogen bond and electrostatic interaction [2]. Poly(ethylene glycol) (PEG) has been widely applied in biotechnological process. In the presence of salt or dextran aqueous two-phase systems are formed, where the top phase is mainly composed by PEG and the bottom phase by dextran or salt solution [8]. These systems work very well in the partitioning of proteins, enzymes and cells, because the biomolecules migrate to the PEG phase [9 11]. The driving force for this migration process seems to be hydrophobic in nature [12]. However, generally the best efciency in partitioning is achieved with PEG of low molecular weight [13,14]. In this work, we investigate the change of the enzymatic activity of G-6-PDH in the absence of salt or dextran, but in the presence three different media: pure Tris HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. We chose G-6-PDH from L. mesenteroides as a model because the mechanism of its catalytic activity is well described in the literature [2]. UV-

spectrophotometry, small angle X-ray scattering (SAXS) with a synchrotron source (LNLS, Brazil) and calorimetric measurements were performed. This systematic study was strongly motivated by the lack of quantitative information [13,14] about the inuence of PEG on the enzyme activity and conformation, although PEG solutions are commonly used in biotechnological process and considered as inert media [9,15,16].

2. Materials and methods

2.1. Materials
Poly(ethylene glycol), PEG, samples with nominal molecular weight of 400 and 4000 Da were purchased from Merck (Darmstadt, FRG). bNicotinamide adenine dinucleotide phosphate (NADP + , N0505), Tris HCl buffer of pH 7.5, glucose-6-phosphate (G-6-P) and glucose-6-phosphate dehydrogenase from the microorganism L. mesenteroides (G-6-PDH, G8529) were obtained from Sigma (St. Louis, USA). The samples were prepared in two different enzyme concentration ranges, as presented in Table 1. The reason for this is the different sensitivity range of each method used, as described below.

2.2. Measurement of enzyme acti6ity

The G-6-PDH activity was measured by spectrophotometric determination with a Beckmann DU 600 Spectrophotometer at 30 C of rate of appearance of reduced NADP + at the wavelength (u ) of 340 nm, according to the method described

Table 1 Reactant concentrations used in each experimental method Method Spectrophotometry SAXS Calorimetry NADP+ 61010 M 61010 M G-6-P 1108 M G-6-PDH 71015 M 1.41010 M 91015 M PEG400 3109 M 5107 M 1109 M PEG4000 31010 M 5108 M 11010 M

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by Bergmeyer [17]. One G-6-PDH unit (U) was dened as the amount of enzyme catalyzing the reduction of 1 mmol of NADP + per min under the assay conditions. The volume and concentration of each reagent mixed in the spectrophotometric cell were the following: 900 ml of 50 mM Tris HCl buffer (pH 7.5), 20 ml of a 10 mM G-6-P solution, 5 ml of a 0.5 mM NADP + solution and 0.34 mg of G-6-PDH. The measurements involving PEG required the addition of either 5 ml of an aqueous solution of PEG400 20 wt.% or 5 ml of an aqueous solution of PEG4000 20 wt.%. The nal enzyme concentration amounted to 7 10 15 M. G-6-PDH concentrations higher than this increase too much the production of NADPH, overloading the detector. The spectrophotometric measurements were performed immediately after the sample preparation and in intervals of 1 h in a total period of 5 h. The enzyme activity degradation was avoided by the storage of the samples in ice bath until measurement. PEG concentration of 20 wt.% in the solutions was chosen because it is close to that used in aqueous two-phase systems for protein partitioning process. The reactants concentration is summarized in Table 1.

20 wt.%. After the fourth titrant addition, the concentrations used in the calorimetric studies are close to those used in the spectrophotomeric measurements (Table 1). The differences between the dilution enthalpy in pure buffer and that in the titration curve in polymer solution are ascribed to the NADP + /PEG binding. Both electrical and chemical calibrations [18] were performed conrming the accuracy of the methodology employed. Since the experimental data must be collected under isothermic conditions, the samples were conditioned in the equipment during 1 h before each titrant addition. A complete set of data took about 5 h for each system.

2.4. Small angle X -ray scattering

SAXS experiments were carried out at National Laboratory of Synchrotron Light (LNLS, Campinas, Brazil) at room temperature of (22 9 1) C, , . Enzyme with radiation wavelength u = 1.608 A samples composed of solutions in the concentration of 7.5 mg protein/ml (Table 1) in absence and presence of PEG were sealed in 1 mm thick acrylic cells, with mylar windows, perpendicular to the incident X-ray beam. It was not possible to measure solutions with concentrations lower than 7.5 mg protein/ml (1.4 10 10 M), i.e., in the same concentration as that used for the spectrophotometric measurements, because the particle volume fraction was too low to give a measurable X-ray scattering (reasonable signal to noise ratio). Freshly prepared samples were measured twice with time acquisition of 20 min each run. The data presented here represent the corresponding average. No radiation damage was observed during the data acquisition. The obtained curves were corrected for detector uniformity (one-dimension position sensitive detector) and proper corrections have been carried out for the decrease of the X-ray beam intensity during the experiment. The parasitic background (buffer solution) was subtracted considering the samples attenuation. No corrections by either slit width or slit length were performed, since these effects are negligibly small on the SAXS curves under the experimental conditions employed in the data collection.

2.3. Calorimetric measurements

Calorimetric measurements were performed using a microcalorimeter model 2277 TAM (Thermal Activity Monitor System, Thermometric AB, Ja rfa lla, Sweden) at 298.15 K, in order to determine the enthalpy of interaction between the different components. The enthalpy changes associated with the interaction between components were assessed by comparison of dilution enthalpies of one of the components in buffer solution with respect to the values for dilution in the presence of the other component. For instance, enthalpy of interaction of NADP + with PEG400 was determined in an experiment that consisted in a series of four consecutive additions (5 ml each) of concentrate NADP + (0.5 mM) to the calorimetric vessel initially containing 3.7 ml of pure Tris HCl solution subtracted from the value obtained from NADP + dilution in 3.7 ml of Tris HCl solution containing 20 ml of PEG400

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It is well known that the SAXS intensity I (q ) of an isotropic solution of monodisperse spheroidal particles of low anisometry (ratio between the largest and the shortest particle axis), is given by [19 22]: I (q ) = kP (q )S (q ) (1) where k = knp(Dz )2V 2; k is a factor related to the instrumental effects, np corresponds to the particle number density; Dz is the electron density contrast between the scattering particle and the medium and V is the scattering particle volume. P (q ) in Eq. (1) is the normalized particle form factor (P (0) = 1) and S (q ) is the interparticle interference factor (q = 4y sin q /u is the scattering vector and 2q is the scattering angle). For low particle number density, S (q ) : 1, so that I (q ) is proportional to P (q ). Several theoretical works have been done to calculate P (q ) for low molecular weight proteins [23 26]. In the current work, we use the CRYSOL program to calculate P (q ) [26]. This software calculates the form factor for macromolecules of a known atomic structure. One can also obtain some information concerning the scattering objects from the experimental curve through the analysis of the distance distribution function p (r ). The scattering intensity I (q ) is described by a Fourier transform of p (r ) [27]: p (r ) =

The Rg value can also be evaluated from the scattering curve at low q range where the intensity obeys the Guinier law [19]: I (q ) = k exp

q 2R 2 g 3

(4)

so that Rg can be directly calculated from the slope of the linear part of ln[I (q )] versus q 2 plot (Guinier plot).

3. Results and discussion

3.1. Inuence of PEG on the enzymatic acti6ity

In order to estimate the inuence of PEG on the enzyme activity, the activity of G-6-PDH was measured in three different media: pure Tris HCl buffer, solution of PEG400 (20 wt.%) in buffer and solution of PEG4000 (20 wt.%) in buffer. In comparison to the enzymatic activity measured in pure buffer, in the presence of PEG400 or PEG4000 the activity of G-6-PDH increased 20%. This increase was found in a set of eight experiments for each medium just after the sample preparation and remained after 5 h. Generally, in the enzyme partitioning process, the activities are measured just after the separation. For this system we veried that over a period of 5 h there is no time effect on the enzymatic activity. Grimonprez and Johansson [14] reported that in the presence of PEG with a molecular weight of 3350 Da the activities of phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase increased 30 and 55%, respectively. The increase of PEG chain length might lead to an increase in the hydrophobicity, as suggested by Forcittini [13]. Following this idea, the structure and the type of active site of a particular enzyme might determine the increase or decrease of the relative activity. In the case of G-6-PDH, the activity increases similarly in the presence of PEG400 or PEG4000, i.e. the chain length seems to have no inuence on the enzymatic activity, at least over 5 h. What is the role played by PEG400 and PEG4000 in this system? Assuming that the enzymatic activity is related to the active site and enzyme conformation, the rst attempt to under-

 &
1 2y 2

I (q )qr sin(qr )dq

(2)

which provides information about the shape of the particle and p (r ) = 0 for r distances larger than the particle maximum dimension Dmax. For isometric objects, as spheres, p (r ) has a maximum around Dmax/2. For anisotropic objects, p (r ) decays linearly at distances between the object crosssection size and its maximum dimension [28]. Further, the p (r ) function also furnishes the value of the particle radius of gyration, Rg, through [27]: 2 Rg= 0 2

& &

Dmax

z (r )2dr Dmax z (r )dr

(3)

In this work, we use the GNOM program [29] to calculate p (r ) from the SAXS curves.

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stand this effect is the determination of the enzyme conformational state changes in the presence of PEG400 or PEG4000 by means of small angle X-ray scattering measurements, as described below.

3.2. Small angle X -ray scattering

G-6-PDH is a dimer of identical subunits of 54 316 Da with two active sites per dimer, which are located in a pocket between the coenzyme binding domain and the b + a domain in each subunit [1]. The dimensions of the crystallized G-6-PDH were determined [2,3] as a = b = 105.71 , , c = 224.31 A , , h = b = 90, k = 120 (according A to the crystallographic structure 1DPG in the RCSB Protein Data Bank [3]). The scattering curves were obtained from G-6-PDH in three different systems: pure buffer, solution of PEG400 20 wt.% and solution of PEG4000 20 wt.%. The results are shown in Fig. 1a and b. , are noisy, The SAXS curves for q \ 0.1 per A because this kind of sample (protein in aqueous solution) presents a very low electron density contrast Dz between the scattering particle and the solvent. Moreover, we are dealing within a diluted regime (small np values) in order to avoid the interparticle interference effects in the curves. It should be remarked, however, that the aim of the current work is to study variations in the tertiary structure of the protein, as a function of PEG molecular weight and this is well reected in the inner q region of the SAXS curves. Slight modications in the ne details of the protein would lead to short-range density uctuations that should be noted in the high q values, but this is not the focus of this work. As one can see from Fig. 1a, the SAXS curves for protein in pure buffer (open triangles) and in the presence of PEG400 (open squares) are identical, showing that, under the experimental conditions employed in these measurements, the enzyme state is similar when dissolved in pure buffer and in PEG400 solution. However, the SAXS curve from PEG4000 is rather different (Fig. 1b), presenting a lower slope value in the inner region. This fact is the rst evidence that the protein/PEG4000 complex has a smaller Rg value

Fig. 1. SAXS curves measured for G-6-PDH in: (a) () pure buffer, ( ) PEG400 solution and (b) () PEG4000 solution, along with the theoretical curve calculated from the dimeric crystallographic structure [3].

than the two other studied samples. This point will be better explored below. With the aim of understanding the different behavior of G-6-PDH in pure buffer and in solution containing PEG400 from that in the presence of PEG4000, we rst calculated the SAXS intensity (P (q ) function) from G-6-PDH using the CRYSOL software [26], by taking into account the dimeric crystallographic structure (1DPG [3]). Fig. 1a and b show the respective P (q ) model (solid line) superimposed to the experimental data. There is a good agreement between the theoretical modeling and the experiment for G-6PDH in the presence of PEG4000, indicating that, in this case, the enzyme assumes a conformation , ). close to the crystalline structure (Rg = 37 A From the P (q ) model a radius of gyration Rg = 40 , is calculated with the assumption of the presA

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, thick hydration layer [26]. Nevertheence of a 3 A less, there is a clear mismatch between the scattering function generated from the crystallographic structure and the experimental curves for G-6PDH dissolved either in pure buffer or in PEG400 solution at low q range (Fig. 1a). This difference was evaluated through the p (r ) and Guinier analyses as follows. The distance distribution functions p (r ) and the corresponding scattering intensities from G-6PDH in PEG400 (similar to buffer) and PEG4000 are shown in Fig. 2a c, respectively, along with the experimental data. The resulting values of I (0), Dmax and Rg (Eq. (3)) from the p (r ) functions are listed in Table 2. It is clear from our results that G-6-PDH dissolved in solution of PEG4000 presents a Rg value similar to that observed for the enzyme state in the crystallization process (dimeric form). For the system composed of G-6-PDH in pure buffer and in the presence of PEG400, the maximum dimension as well as the radius of gyration are almost twice as large (Table 2), evidencing an alteration in the scattering object. The value of I (0 ) increases by a factor of about 4 in relation to that observed for protein/PEG4000 complex (enzyme dimeric conguration) (Table 2). This indicates a further aggregation phenomenon. Therefore, a more complex agglomerated structure must be formed in pure buffer and in the presence of PEG400 than in PEG4000. Finally, the Guiniers plots are shown in Fig. 3 in the small q region. The Rg values are included in Table 2 for comparison. G-6-PDH in the presence of PEG4000 presents a Guinier region with , (within the Guinier limit qRg B 1) [19], Rg = 37 A in agreement with that obtained from the structure model (1DPG) and p (r ) analysis. On the other hand, the curve corresponding to the pure protein (and with PEG400, data not shown) does not exhibit a clear linear range within the same interval, indicating a polydisperse system. It means that different aggregates of G-6-PDH are formed in pure buffer solution and in the presence , (straight line in of PEG400. A value of Rg $ 70 A the inner q region, Fig. 3) is compatible to that extracted from p (r ) analysis.

Fig. 2. (a) Distance distribution functions p (r ) obtained for G6-PDH in PEG400 (solid line) and PEG4000 (dotted line). Corresponding scattering intensities I (q ) obtained from G-6PDH in (b) PEG400 (similar to pure buffer) and in (c) PEG4000, along with the experimental data.

S.M. Pancera et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 291 300 Table 2 Radius of gyration (Rg) calculated by means of Solvent
CRYSOL ,) Rg (A

297

CRYSOL

software p (r ) function ,) RgEq. (3) (A I(0) 0.02386 0.02386 0.005575 ,) Dmax (A 220 220 120

,) R Guinier (A g

Buffer PEG400 PEG4000

40

70 70 37

74 74 43

Guinier analysis (Eq. (4)) and from Eq. (3); particle maximum dimension Dmax and I (0) evaluated from p (r ) function for G-6-PDH in buffer, 20 wt.% PEG400 solution and 20 wt.% PEG4000 solution.

It should be remarked that, in the SAXS curve for the enzyme in pure buffer and in presence of PEG400 (Figs. 1 3), the evaluated maximum dimension is related to the maximum slope in the accessible q range due to the experimental resolution of our set-up. In this context, larger complexes of aggregated proteins might exist in solution but, if so, they could not be detected by this technique. Therefore, the data analysis obtained from SAXS curves showed that PEG molecular weight has an effect on the enzyme conformation, in such a way that PEG4000 reduces non-specic aggregation in solutions of G-6-PDH.

3.3. Calorimetric measurement

The catalytic mechanism for the enzymatic activity of G-6-PDH on the oxidation of glucose-6phosphate was recently proposed by Cosgrove and co-workers [2], as shown in Fig. 4. The amino acid His-240 acts like a general base abstracting a proton from the C1-OH. In the transition state, a positive charge is formed in His-240. This positive charge is stabilized by the carboxylate group of Asp-177. That is the reason for the formation of the so-called catalytic dyad. The next step is the transfer of the hydride from C1 to C4 position of the pyridine ring of NADP + , yielding the reduced form NADPH. They also showed that the amino acid His-178 is an important residue because it binds to the phosphate moiety of G-6-P base by hydrogen bonding and electrostatic interaction. The existence of specic interactions between PEG and the co-enzyme NADP + or the enzyme

G-6-PDH was investigated by means of calorimetry. The experiments were performed in order to verify whether there is any enthalpy change due to these interactions. For this purpose, the enthalpies of dilution of enzyme and co-enzyme were determined in pure buffer and in the presence of either PEG400 or PEG4000. Any occurring interaction would lead to different values of dilution enthalpy and upon subtraction from the reference value for dilutions in buffer, the enthalpy for the interaction, DHint, between PEG and enzyme or co-enzyme would be obtained [30,31]. The results obtained for the systems studied here are summarized in Table 3. They show that the dilution of NADP + in these media was always exothermic and no dependence of DHdil on the NADP + concentration was observed. Moreover, they indicate that the interactions between NADP + and PEG solutions are enthalpically more favorable than those between NADP + and pure buffer,

Fig. 3. Guiniers plots from SAXS curves for G-6-PDH in ( ) pure buffer (or PEG400) and () PEG4000.

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Fig. 4. Schematic representation of the catalytic mechanism proposed by Cosgrove [2] and co-workers. (A) Transition state, (B) reaction products.

suggesting a reason for the enhancement of enzymatic activity in PEG400 or PEG4000 in comparison to pure buffer over a period of 5 h. Another interesting nding is that the values of DHdil obtained for NADP + dissolved in solution of PEG4000 are more exothermic that those found for PEG400, 99.3 and 81.9 kJ/mol, respectively. An exothermic overall interaction implies that more energetic interactions are formed upon NADP + addition to PEG solutions. Considering the chemical nature of species involved and that water is present in excess (as solvent), it is likely that hydrogen bonding plays a central role among these interactions. However, one has to bear in mind that these enthalpies reect the balance between formed and broken interactions, which is quite complex and subtle, as we recently showed for the interaction between PEG3350 and different electrolytes in aqueous solutions [32]. One difference between PEG 400 and 4000 is the magnitude of the end-group (hydroxyl) contribution to their solution behavior. The transition from a glycolic to a polyether behavior for PEG occurs between molecular weights of 2000 and 3000 Da. Hence, PEG400 would display a signicant contribution from interactions arising from its hydroxyl groups, whereas for PEG4000 only (CH2CH2O) units play an important role. We believe such a discrimination is more informative

than the common statement that PEG becomes more hydrophobic as its molecular weight increases, as put forward by Forcittini [13], for instance. Such a difference must be related to the different dilution enthalpies displayed by NADP + in the different PEG, but it is not clear exactly which kind of interaction(s) is responsible. A systematic study exploring the effect of PEG chain length on the interaction with NADP + will be carried out. Contrary to NADP + , G-6-PDH showed no interactions with the media studied here, since all DHdil values amounted to zero (Table 3). However, since the amounts of enzyme used in the experiments are quite low, caution should be taken in ruling out. These ndings would conrm the statement often reported [9,15,16] that PEG act as an inert polymer, since it does not interact with proteins. On the other hand, the spectrophotometric measurements showed an increase of 20% in the enzymatic activity, corroborating with the idea that this increase is caused by the favorable interactions between NADP + and PEG, at least in this concentration range.

4. Conclusions PEG is commonly referred as an inert polymer in biotechnological applications. Contrarily to

S.M. Pancera et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 291 300 Table 3 The enthalpic change upon dilution Titrant DHdil/TrisHCl 72.6 9 0.8 0 DHdil /PEG400 solution 81.9 9 0.9 0 DHint with PEG400 DHdil/PEG4000 solution 99.3 9 0.9 0 DHint with PEG4000

299

NADP+ G-6-PDH

81.9(72.6) =9.3 0

99.3(72.6) =26.7 0

DHdil (kJ/mol), and the difference between DHdil (kJ/mol) measured for the titrant (NADP+ or G-6-PDH) in PEG solution and that measured in buffer, DHint (kJ/mol), obtained for NADP+ and G-6-PDH (for more details, see text).

this statement, in this work we show quantitative results in two different enzyme concentration ranges, which reveal a signicant effect of PEG on the enzymatic activity and on the enzyme state in solution. In a very low G-6-PDH concentration (7 10 15 M), it was shown by spectrophotometric and calorimetric measurements that PEG increases the enzymatic activity due to favorable interactions between the co-enzyme NADP + and PEG and not due to the interactions between G-6-PDH and PEG. This is a very important nding, since NADP + is common in many biological processes responsible for the ATP production. On the other hand, increasing the G-6-PDH concentration to 1.4 10 10 M, different states of enzyme aggregation were evidenced by SAXS. In the presence of PEG4000, a monodisperse system composed of protein in the native crystallographic structure (dimeric conguration) was observed, whereas in the presence of PEG400 a polydisperse system formed by greater agglomerates was evidenced, similar to that observed in pure buffer. We propose that different effects of PEG 400 and 4000 be ascribed to the existence of signicant contribution of hydroxyl groups for the solution behavior of the former, while, for the latter, these end-group contributions are not relevant. To our knowledge it is the rst time that the inuence of PEG on the enzyme and co-enzyme behavior was investigated. Other systematic studies focusing on the inuence of PEG over a broader range of molecular weight and concentration, as well as of the enzyme concentration on the structural conformation and enzymatic activity will be carried out.

Acknowledgements The authors thank the National Laboratory of Synchrotron Light (LNLS, Campinas, Brazil) for the use of their facilities. This work was supported by research grants from FAPESP (Grant Nr. 97/13070-2) and CNPq. R.I. also acknowledges Pronex/MCT/CNPq.

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