Lipid Interfaces

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Hyaluronidase Behavior at the Air/Liquid and Air/Lipid Interfaces and Improved Enzymatic Activity by Its Immobilization in a Biomembrane Model Douglas Santos Monteiro,† Thatyane Morimoto Nobre,‡ and Maria Elisabete Darbello Zaniquelli* Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeir~ao Preto, Universidade de S~ao Paulo, 14040-901 Ribeir~ao Preto, SP, Brazil ABSTRACT: Bovine testicular hyaluronidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid catalytic hydrolysis, was successfully immobilized on LangmuirBlodgett films prepared with the sodium salt of dihexadecylphosphoric acid, (DHPZn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein adsorption at the airliquid interface by means of pendant drop shape analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 μg L1 attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at πf0. The surface pressure (π) area curves obtained for BT-HAase and mixed DPPC BT-HAase monolayers reveal the presence of the enzyme at the airlipid interface up to 45 mN m1. Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.

’ INTRODUCTION Hyaluronan (hyaluronic Acid, HA) is a high molar weight heteropolysaccharide, which is linear and alternates residues of D-glucuronic acid and N-acetylglucosamine, in a structure of poly[β(1f3)-2-acetamido-2-deoxy-D-glucose-(1f4)-β-D-glucopyranosyluronic acid] (Figure 1). These nonsulfated glycosaminoglycans are present in prokaryotic as well as eukaryotic organisms. In vertebrates it can be found as an extracellular matrix component of skin and connective tissue. Moreover, HA is also involved in many biological events including fertilization, growth and metastasis of tumor cells, differentiation and migration of cells, embryonic development, wound healing, and inflammation.1 Hyaluronidase (HAase) is a term created for a class of enzymes responsible for randomly HA catalytic hydrolysis,2,3 and which are classified according to the HA degradation mechanism, the one used in this work being a hyaluronate 4-glycanohydrolase (hyaluronoglucosaminidase, EC 3.2.1.35).4 It is an important enzyme in biological and biochemical sciences which is found in several mammalian tissues and body fluids such as serum, synovial fluid, and urine.57 Furthermore, HAase is described in poisonous animals such as snakes and scorpions,8,9 where it supposedly acts as a spreading factor, degrading HA and permitting diffusion of venom in the victim, although HAase is nontoxic by itself.10 Despite its broad presence and functions in different organisms, it is recognized10,11 that this class of enzyme and its substrate has been neglected.4,11 More recently, efforts have r 2011 American Chemical Society

been made toward understanding HAase surface physical chemistry properties, as well as its interactions with biomembrane mimetic models as phospholipid Langmuir monolayers and LangmuirBlodgett (LB) films.1214 Studies of HAase in biomimetic membrane or lipid template are relevant on the point of view of fundamental investigation aiming to understand the surface activity features of this enzyme and its interaction with cell surface. Moreover, nanotechnological applications of HAase, mainly as a biosensor, have arisen as an interesting system, since researchers correlated some type of cancer with the anomalous expression of hyaluronan.1518 In this sense, LB films can render useful templates for immobilization of biomacromolecules and biosensor preparation.1922 Furthermore, besides these potential applications, the possibility of molecular packing and lipid composition control for the Langmuir monolayers, as well as defined thickness and sequence of LB films, they are extensively used as biomembrane models for molecular recognition studies2325 and enzyme catalytic performance at the molecular level.2628 The aim of this study is to investigate the enzymatic activity of HAase extracted from bovine tests, BT-HAase, when immobilized in LangmuirBlodgett films, which is relevant for the development of a biosensor fabrication based on HAase function. Received: November 11, 2010 Revised: January 27, 2011 Published: April 05, 2011 4801

dx.doi.org/10.1021/jp110795d | J. Phys. Chem. B 2011, 115, 4801–4809

The Journal of Physical Chemistry B

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Figure 1. Structure of hyaluronic acid (HA).

From the best of our knowledge, no investigations about HAase immobilization and its enzymatic activity in LB films have been reported in literature, and the results shown in this paper indicate a strong increase in the biological activity for the enzyme entrapped on the nanostructured film. For this purpose, we have first investigated the adsorption of BT-HAase at liquidair interface and liquidlipid interface in order to choose the best conditions for the enzyme immobilization as judged by the deposited mass of the proteolipid LB film by means of the quartz crystal microbalance. Finally, the HAase enzymatic activity immobilized in LB film is evaluated and compared to the one in homogeneous media (free enzyme in solution).

’ EXPERIMENTAL METHODS Materials. Bovine testicular HAase (BT-HAase) is a tetrameric29 60 KDa glycoprotein30 (Sigma, IS type, lyophilized powder, EC 3.2.1.35). Hyaluronic acid potassium salt from human umbilical, the phospholipids—dipalmitoylphosphatidylcholine sodium salt (DPPC), dihexadecyl phosphate sodium salt (DHP), N-acetyl-Dglucosamine (NAG, purity > 99%), and p-dimethylaminobenzaldehyde (DMAB, purity 99%)—were also purchased from Sigma. Water was supplied by a Milli-Q system (resistivity, 18.2 MΩ cm; surface tension, 72.8 mN m1). Ethanol-stabilized chloroform was chromatographic grade (J. T. Baker). Other reagents were of the highest purity commercially available. Adsorption Kinetics. Adsorption kinetics of BT-HAase at the airliquid interface was followed by means of surface tension (γ) changes with time, using the pendant drop method and shape profile analysis in an automatic tensiometer (OCA-20, Dataphysics Instruments, Filderstadt, Germany). A drop of BT-HAase solution was formed at the tip of a needle, and a freshly created surface was provided by an injection system. A movie tracking in intervals as short as 0.02 s was recorded by a CCD camera, managed by the tensiometer SCA20 software. Drop parameters as surface area and volume can be obtained, and the surface tension was calculated by the adjustment of drop coordinates to the YoungLaplace equation. The surface pressure variation (Δπ = γ(t) γ0), taking γ0 as the value measured for the airaqueous buffer solution interface, was recorded for BTHAase adsorption at a bare airbuffer (phosphate pH 5.3; NaCl, 0.15 M) interface. Time-dependent effects during the adsorption of BT-HAase at DPPC monolayers were followed at a 216 cm2 Langmuir trough (Insite, Ribeir~ao Preto, Brazil) by the spreading of DPPC dissolved in 1 mmol L1 chloroform solution. Monolayers were spread on pure water subphase or phosphate buffer (0.1 M, pH 5.3; NaCl, 0.15 M) aqueous solution, to an initial area ca. 110 Å2 molecule1 and compressed at 5 Å2 molecule1 s1, to obtain the entire πA curve or up to a desired initial surface pressure (πi), as measured by the Wilhelmy plate method. Surface pressure was monitored as a function of time to ensure monolayer stability. Then, small aliquots of BT-HAase were injected

into the subphase, close to the interface to render a 5 μg L1 final concentration of the enzyme in the trough, and surface pressure variations were continuously recorded until constant values were reached. LB Films. Initially, Langmuir monolayers were prepared by spreading an appropriated volume of 1 mmol L1 DHP chloroform solution over a Zn2þ 0.1 mol L1 subphase. For LB deposition, the surface pressure was kept constant at 30 mN m1 and deposited in a withdrawalinsertingwithdrawal cycle, resulting in a hydrophobic three-layer Y-type LB film. The solid support was quartz crystal gold coated, for QCM measurements, or quartz, for enzymatic activity determination. This three-layer film was dried in a desiccator for at least a half-hour and used as a template for the immobilization of BT-HAase. After that, two different procedures were adopted for the enzyme incorporation into the film. In the first one, the DPPC monolayer at 30 mN m1 was transferred onto DHP hydrophobic Y-type film. After deposition, the new (DHPZn2þ)3DPPC film was immersed into a 5 μg mL1 BT-HAase solution to promote the adsorption of the enzyme from the solution. After being dried, these films were rinsed by immersion in pure water for 5 min. In parallel, (DHPZn2þ)3DPPC films without any protein content were submitted to the same rinse step as a control for BT-HAase mass determination. In the second methodology, DPPC monolayers were compressed to a desired π and the barrier was stopped, allowing the monolayer relaxation process. After the equilibrium was reached, 1.0 mL of BT-HAase was injected into the subphase to render a final protein concentration of 5 μg mL1. The surface pressure was recorded continuously until no variation was detected. Then a BT-HAaseDPPC fourth layer was deposited on a previous (DHPZn2þ)3 LB film. The proteolipid LB film was rinsed for 30 min, in the same solution used for the enzymatic activity assays. This process was repeated three times. After each step of the film assembling, including rinse, the deposited mass was assessed by nanogravimetry using quartz crystal microbalance technique (QCM). For these measurements, the films were deposited onto AT-cut quartz crystal coated with Au (active area, 0.662 cm2; fundamental frequency, ca. 10 MHz) connected to a resonator (ICM lever oscillator 35366, International Crystal Manufacturing, Oklahoma, OK, USA) and to a frequency meter (Lutron FC-2700 TCXO). Enzymatic Assays. For BT-HAase enzymatic assay, the fluorimetric MorganElson method described by Takahashi et al.31 was adopted with slight modifications concerning the incubation time of 100 or 110 min, instead of 20 min used by these authors. In this method the hydrolysis of HA in phosphate buffer (pH 6.0) is carried out in the presence of HAase. The HA hydrolysis product, N-acetyl-D-glucosamine (NAG), is complexed with p-(dimethylamino)benzaldehyde (DMAB) in tetraborate solutions, pH 10.5, rendering a fluorescent compound, which is detected by emission spectroscopy (excitation and emission wavelengths of 545 and 604 nm, respectively; 4802

dx.doi.org/10.1021/jp110795d |J. Phys. Chem. B 2011, 115, 4801–4809


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spectrofluorometer Spex Triax 550). The enzymatic activity (EA) was expressed in units of HAase activity, which corresponded to 1 [μmol of NAG/(mg of HAase)]/(min of reaction). To construct a calibration curve for BT-HAase activity determination, predefined amounts of NAG in solution were reacted with DMAB in tetraborate solutions, and the emission of the fluorescent colored product was plotted as a function of NAG concentration. The enzymatic activity in heterogeneous medium was carried out following the same methodology as described above, by immersing HAase-DPPC LB films in 1.5 mg mL1 HA solution. Then, aliquots of substrate reacted solution were submitted to MorganElson reaction in order to obtain the fluorescent product, so the NAG concentration has been determined by comparison with the standard calibration curve. The temperature for adsorption and Langmuir monolayers studies was 24.0 ( 0.5 °C. The enzymatic essays were carried out at 37 °C, and QCM and emission readings were taken at room temperature, 25 ( 1 °C

’ RESULTS AND DISCUSSION BT-HAase Surface Activity. Human hyaluronidase (h-HAase)

is considerably important since it degrades HA and it is present in larger concentrations in cancer patients. Structural information was first available for the bee venom hyaluronidase,32 but it shows a sequence identity of only 32% with h-HAase, which was reported only recently.33 Nevertheless, the homology between h-HAase and BT-HAase, used in the present study, is 64%,34 and the results here described should be relevant for future applications of h-HAase.35,36 A description of surface activity of BT-HAase is already found in the literature,12,14 using Langmuir trough experiments. To characterize the surface activity of this enzyme, we have monitored surface pressure variations for BT-HAase homogeneous solution at different concentrations in freshly formed pendant drops (Figure 2). These data help one to choose a protein concentration to study the proteinphospholipid interaction and give support to interpret the data that have arisen from this study. For these experiments, the solution is homogeneous and the volume (∼1315 μL) is quite small compared to the one used in the Langmuir trough (135 mL). Therefore surface activity effects come only due to diffusion from the subsurface to the interface, as the concept introduced by Ward and Tordai,37 and not from possible artifacts evolving convection and mixture in the bulk. In this sense, it is not peculiar that one could find different lag or induction times t*, when the measurements are carried out at the Langmuir trough by means of protein injection experiments. t* corresponds to the period of time from zero (surface just created) to the one when π just begins to differ from zero, and it is observed38 because proteins adsorbed at interfaces can assume different states associated with different molar surface areas (ωi). At low surface pressures the adsorption layer consists of molecules requiring a maximum surface area. In other words, at low π the protein should be unfolded. From the adsorption isotherm (eq 1) it is possible to see that if ω is large, π is low and, in this sense, π will reach values different from zero only when the coverage (θi = Γiωi) exceeds roughly 0.10.2.39 π ¼ ðRT=ωÞ½lnð1 ΓωÞ ael ðΓÞ2 ω2

Figure 2. Adsorption kinetics of BT-HAase at the bufferliquid interface as measured from surface pressure variations at freshly formed surfaces of BT-HAase pendant drops in different concentrations: (boxes) 10.0, (circles) 15.0, (triangles) 20.0, and (diamonds) 30 μg mL1. Temperature: 24.0 ( 0.5 °C.

From Figure 2 it is possible to determine t*. They are respectively 5.8, 3.2, and 1.7 min for protein bulk concentrations of 10, 15, and 20 μg mL1, respectively. For 30 μg mL1 we could not detect t* within the accuracy of our experiments. On the other hand, for the protein concentration of 5 μg mL1 one could not observe any π variation in the adsorption period of the experiment. If the adsorption is governed by diffusion, the relationship c2t* ∼ constant should hold38 at the low π regime. In the case of BTHAase we found the (3.9 ( 0.4) 104 (μg mL1)2 s value for this product. Therefore we can say that the mechanism is predominantly diffusive and one can estimate t* for the protein concentration of 5 μg mL1 close to a half-hour. In a previous report12 using experiments at the Langmuir trough and BT-HAase concentration of 1.4 μg mL1, the authors found a lag time of about 16 min, underestimated if one considers the adsorption of the same protein as the one used in this work. As the protein was spread on or injected into the subphase of the Langmuir trough, it is most likely the cause of discrepancy between the results was the concentration gradient, which is larger in the Langmuir trough for the same final bulk concentration. In that case the solution is not homogeneous when the adsorption is started. Also, for comparison, with other native globular proteins the observed t* = 10 min, with a Δπ = (πeq πin) ∼ 17 mN m1,40 whereas for bovine serum albumin (BSA; Mr close to BT-HAase) no lag time is observed for 10 μg mL1.40 The hydrophobicity of BSA, as measured using fluorescent probes, is between ∼15- and 50-fold that of ovoalbumin (OVA), depending on the utilized probes.41 One should also consider their respective flexibilities. Thus, it is most likely BT-HAase used in this study may present a hydrophobic moiety upon adsorption whose relative hydrophobicity is between OVA and BSA, being closer to BSA. Having the induction times, it is also possible to estimate the total surface concentration at the onset of a surface pressure increase, the critical adsorption, Γ*πf0, denoted simply as Γ*, from the bulk concentration, co, by using the eq 2 and assuming a diffusion coefficient, D, of 6 107 cm2 s1 (for comparison with D for bovine serum albumin, 64 kDa).

ð1Þ

where Γ = Σi Γi (the total adsorption of the protein in all states) and ael is a term that takes into consideration the contribution of the electrical double layer to the surface pressure of the adsorption layer.

Γ ¼ 2co ðDt =πÞ1=2

ð2Þ

For the highest bulk concentration of BT-HAase, 20 μg mL1, Γ* = 0.006 mg m2, whereas it is about 1.1 mg m2, for 4803



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ovoalbumin (Mr, 45 kDa) at a bulk concentration of 100 μg mL1. Moreover, the calculated Γ* for BT-HAase decreases as the bulk concentration increases (Table 1) for the concentration range of 530 μg mL1. This apparent inconsistency is easily explained, if one considers that the surface excess calculated from eq 2 depends on both co and t*, and as the bulk concentration increases, the induction time decreases. Therefore the differences are related to the different periods of time the adsorption is taking place. On the other hand, at a fixed surface pressure, such as for example πf0 or at the onset of π, there is a distribution of different states Γι* as a function of the surface area ωi covered by the protein. Moreover, it is shown39 that, in spite of different states possibly being adsorbed, some are more likely than others. However at t*, Γ = Γ*, and in this sense it is most likely that for 5 μg mL1 the most probable conformation of the protein should be the one which occupies the smallest area at π f 0, in the concentration range of 530 μg mL1, since Γ* is the highest for the same unity area. Interaction of BT-HAase with DPPC Monolayers. From the results of adsorption kinetics of BT-HAase on a clean airbuffer interface, one chooses to work with a BT-HAase final bulk concentration of 5 μg mL1 to investigate the interaction with DPPC. The choice of DPPC to build our membrane model was due to the large presence of phosphatidylcholine derivatives in biomembranes. Characterization of BT-HAase insertion at different DPPC lipid packing is presented in Figure 3A for experiments using the Langmuir trough. The surface pressure, π, was monitored as a function of time for different surface pressures, πi, in order to allow the monolayer relaxation and attest the Table 1. Estimation of the Total Surface Concentration, Γ*πf0, from the Bulk Concentration, at t = t* [BT-HAase] (μg mL1) t* (min)

5 (26)

10 5.8

15 3.2

20 1.7

Γ*πf0 (ng cm2)

2.5

1.1

0.9

0.6

monolayer stability (data not shown). When π reached a constant value, the protein was injected into the subphase, close to the interface. An immediate increase in the recorded values of π was observed, indicating no induction time for BT-HAase adsorption at the DPPC monolayer under these conditions. A plateau is observed for all initial conditions, indicating that adsorption equilibrium is attained at a certain πeq. In Figure 3B it is possible to analyze the dependence of surface pressure variation, Δπ = πeq πi, with πi. In a general way, Δπ decreased with increasing πi and Δπ tends to zero when πi is just above 30.3 mN m1. Figure 4A shows the πA hysteresis cycle measured for the DPPC monolayer and the compression of a DPPC monolayer after BT-HAase has been injected at πi = 20 mN m1, followed by a waiting time of 3.5 h and expansion up to the maximum trough area. The πi value of 20 mN m1 was chosen in order to approach the lipid packing of a cell membrane and at the same time for being below the πi value for which Δπ = 0. For pure DPPC monolayer, the obtained isotherm is the one expected from previous results found in the literature.42 There is a liquid-expanded to liquid-condensed transition in the range of ∼65 to 45 Å2/molecule, and the minimum molecular area is observed at 46 Å2. Moreover, Figure 4 indicates no hysteresis for the DPPC monolayer. The addition of HAase affected the DPPC film packing, since, upon decompression of the monolayer, the surface pressure was 7 mN m1 (and not zero, as measured for pure DPPC monolayer at the same trough area). The subsequent compression clearly indicates the effect of the BT-HAase onto the lipid monolayer, since the LE-LC HAase transition is extinguished, and the isotherm is shifted to larger molecular areas. The two πA isotherms, for pure DPPC and for DPPCBT-HAase, intercept only at π = 45 mN m1, indicating that BT-HAase was not completely expelled from the interface up to this surface pressure, most likely due to hydrophobic interactions with the monolayer, as previously observed for other proteins.43 Therefore, at the π range of 2530 mN m1, correspondent to the lipid packing of a biomembrane,44 the

Figure 3. (A) Surface pressure variation (Δπ) caused by the injection of BT-HAase under the subphase of DPPC monolayers at controlled temperature of 24.0 ( 0.5 °C at different initial surface pressures (πi) at the Langmuir trough: (triangles pointing up) 8.1, (triangles pointing down) 20.0, (circles) 25.0, and (boxes) 30.0 mN m1. (B) Surface pressure variations (Δπ) versus initial surface pressure of DPPC monolayer (πi). 4804



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Figure 4. (A) Comparison of DPPC isotherms at (triangles) first and (circles) second compression of DPPC monolayer; (boxes) compression of DPPC monolayer after BT-HAase injection at 20 mN m1 followed by expansion up to the maximum trough area. (B) Compressional moduli (Cs1) as a function of surface pressure for (circles) DPPC second compression and (boxes) DPPC-HAase monolayer. Temperature 24 ( 0.5 °C.

enzyme still remains at the DPPC film. It is interesting to observe that some authors call the value of πi for which Δπ tends to zero as the exclusion surface pressure, but this is not true when we have a mixed monolayer for which the surface pressure does not correspond to the surface of the pure phospholipid monolayer at the same molecular area. In addition, the surface compressional modulus or equilibrium dilatational surface elasticity modulus, Cs1 = (∂π/∂ ln A)T, were calculated and presented in Figure 4B as a function of surface pressure for both isotherms, pure DPPC and mixed DPPCBT-HAase. The coincidence of Cs1 for π > 43 mN m1 for both isotherms clearly indicate the displacement of the protein from the interface above 43 mN m1, and the available area at the interface and relative partition of lipid and protein between bulk and surface should be the reason for that. Together the results presented in Figures 3 and 4 attest to the interaction between BT-HAase and DPPC, contrary to the interpretation of Belem-Gonc- alves and co-workers, who erroneously concluded that the decrease in Cs1 with the addition of HAase would represent a protein desorption during compression. In fact, it is well-known from literature42,45,46 that many proteins which interact with phospholipid monolayers decrease the equilibrium as well as dynamic surface elasticities (measured by different techniques), and this is actually a clear indicative of protein incorporation within the lipid monolayer. On the other hand, as from our results, at the protein desorption πdes, the surface elasticity is equal to the pure lipid. The observed decrease in Cs1 is a consequence of an increase in the fluidity of the lipid film, since the incorporation of the protein makes harder the lipid packing. Nevertheless, these results also suggest that the insertion of HAase is difficult at higher DPPC surface packing, which is in agreement with other results of interaction between enzyme or peptides and lipid monolayers22,43,47 but is not relevant if the aim is to investigate the interaction with a biomembrane model since in biological medium the cell surface never reaches such

Table 2. Deposited Mass for Each Step of the Immobilization of BT-HAase in the Phospholipid LB Film by Using Protein Adsorption from Aqueous Solution mass (ng)

mass (ng)

DHP/Zn2þ (three layers) 470.8 ( 19.6 after 1st rinse step 398.6 ( 51.7 108.5 ( 3.9 after 2nd rinse step 266.1 ( 52.6 DPPC (one layera) DPPC þ BT-HAase

326.5 ( 37.7

a

Determined from a control experiment when the DPPC layer was dried in a desiccator for 1 h.

high values. In addition, this previous study provides information about possible conditions for immobilizing BT-HAase in DPPC LB films. BT-HAase Immobilization in LB Films. As multilayers of DPPC are not easy to build up, LB films were prepared by the deposition of three layers of DHP/Zn2þ at 30 mN m1 as a template. The choice for this matrix is mainly due to the high transfer ratio (TR) for this phospholipid at this condition and considerable intermolecular interaction between successive layers, providing good film stability. A TR value close to 1 indicates that most likely the surface of the solid support is completely covered by the LB film, producing a homogeneous coating. However DHP itself would not be a suitable matrix for enzymes due to its high surface packing. Moreover PCs should be more appropriate to biomacromolecules since these lipids are the main component of biomembranes. With these three layers, the outmost layer of the Y-type LB film was hydrophobic, facilitating the deposition of an end-capping layer of DPPC or DPPCprotein. The average deposited masses for each step of the proteolipid film assembling were determined by QCM, and the values are presented in Tables 2 and 3. The average mass per layer of DHP/ Zn2þ LB template was about 153 ng, in agreement with that previous reported in the literature.48 Rinsing this matrix film does 4805



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Table 3. Deposited Mass for Each Step of the Immobilization of BT-HAase as a Proteolipid Filma mass (ng)

mass (ng)

DHP/Zn2þ (three layers)

452 ( 28

DPPC þ BT-HAase after rinse step

358 ( 14

DPPC (one layerb)

126 ( 9

BT-HAase

243 ( 30

DPPC þ BT-HAase

369 ( 28

The protein is deposited at 30 mN m1 as a mixed monolayer with DPPC formed at the Langmuir trough. b Value estimated from the π-A curve for the mixed monolayer. a

not produce any change in the crystal frequency. As already described, we have used two methodologies for the enzyme immobilization. In the first one the DPPC monolayer was transferred as the fourth layer, and immediately after the deposition, the film was immersed in a 5 μg mL1 BT-HAase aqueous solution. Experiments were carried out in parallel, drying the films with the fourth layer to determine the DPPC deposited mass, rendering a mass per layer of about 109 ng. The deposited mass was ∼60% of that expected from the molecular area of DPPC at the deposition surface pressure (Figure 4A) and the active area of the piezoelectric crystal. Moreover, only 20% of the DPPC deposited mass remains when this film is rinsed with water. Nevertheless, immersing the (DHP)3DPPC LB films without rinsing in the BT-HAase aqueous solution for 5 min, following drying in a desiccator for 1 h, the obtained mass was around 399 ng (Table 2). Rinsing this film has produced a change of 33% in the deposited mass, indicating the protein preserves the DPPC within the film. Therefore, the minimum deposited BTHAase, subtracting the mass for DPPC is about 290 ng, equivalent to a surface density of 7.4 pmol cm2. The second alternative for the immobilization of BT-HAase was to inject the protein in the subphase of the DPPC Langmuir monolayer (to render a protein concentration of 5 μg mL1), at 20 mN m1. This π value was chosen because the equilibrium surface pressure for this condition was 25 mN m1 (see Figure 3). Therefore, to deposit the proteolipid film at 30 mN m1, a large compression of the surface was not necessary. The LB deposition was started ca. 3.5 h after enzyme injection, as a certain guarantee for better BT-HAase distribution under lipidic monolayer. After drying we obtained a total mass of 369 ( 28 ng (Table 3). From the πA isotherm (Figure 4) the expected mass of DPPC in the mixed monolayer transferred to the active area of the crystal is about 126 ng. Therefore the estimated protein mass in this case was 243 ( 30 ng, correspondent to a surface density of 6.1 pmol cm2 or 2700 Å2 molecule1. The surface density obtained in this work is in the range of that found for BSA in different kinds of interfaces.49 Moreover if we suppose that the area occupied per protein molecule at the LB film is the same as the one it occupies at the airliquid interface, one can estimate that, for the difference in area, ΔA, at the deposition surface pressure, 30 mN m1, obtained for the mixed DPPCBTHAase and pure DPPC monolayers (ΔA = 20.7 Å2 molecule1, from Figure 4A), the DPPC:BT-HAase is around 135:1. Of course this is a coarse estimative since there is no guarantee the surface area of the enzyme in the two kinds of interfaces is the same. Comparing the two methodologies, the film built by the LangmuirBlodgett technique resulted in better reproducibility, as observed from the standard deviation values presented in Tables 2 and 3. Furthermore, this immobilization rendered films with higher stability, since the BT-HAase mass remaned almost constant after the rinse step. Furthermore, BT-HAase seems to

stabilize DPPC LB film, since only DPPC film was not resistant to the rinse step whereas in DPPCHAase film the mass remains nearly constant (Table 3, see entries “DPPC þ BT-HAase” and “DPPC þ BT-HAase after rinse step”). Therefore we choose this methodology to attest to the enzymatic activity of the immobilized BT-HAase. BT-HAase Enzymatic Activity. The enzymatic activity of BTHAase was achieved from its ability of promoting the cleavage of HA, producing NAG. Because the protein amount in the LB film was in nanoscale, we chose a highly sensitive method for NAG determination. In this sense, the fluorimetric MorganElson assay, modified and optimized for detection limit reduction,31 was chosen, despite the several HAase enzymatic activity determination methods found in literature. Fluorescence spectra of NAG standard solutions and BT-HAase cleavage product (Figure 5A,C, respectively) allowed us to construct reliable calibration curves, of fluorescence intensity versus concentration, from known amounts of NAG and BT-HAase, respectively (Figure 5B,D), with linear correlation coefficient higher than 0.99. The fluorescence intensities were corrected by subtracting the signal of the blank from the signal gotten from the samples. The blank consisted of a solution of HA in the absence of BTHAase, submitted to the same reactions. The enzymatic activity, EA, in homogeneous medium was calculated by the ratio between the slopes of BT-HAase and NAG standard calibration curves, divided by the incubation time (110 min). For the heterogeneous medium the LB film (deposited on piezoelectric crystals) with the immobilized BT-HAase was immersed in the HA solution and the procedure was exactly the same as that for homogeneous medium regarding the reaction and times. From the calibration curve measured at the same day emission detected for our samples one could determine the produced NAG amount. By means of QCM technique one could determine the amount of immobilized enzyme and calculate the mean EA value. The measurements were carried out in triplicate. The EA values are shown in Table 4 for the homogeneous medium and BT-HAase immobilized in LB phospholipid films. The results for EA of BT-HAase in homogeneous media indicate that each milligram of BT-HAase produced ca. 0.04 μmol of NAG per minute. Under the same experimental conditions, BT-HAase immobilized on LB films rendered an enzymatic activity 22-fold higher than the free enzyme in solution. After being used in the enzymatic reaction, the BT-HAaselipid film was rinsed and kept in desiccator for 12 h, and a new catalysis experiment was performed. The activity was maintained to approximately 45% related to the film used for the first time (Table 4), indicating the preservation of the enzyme within the LB film. It is interesting to observe that even with this reduction the EA for the immobilized enzyme still compares to the one measured in homogeneous medium. This increased activity for immobilized enzymes in LB matrix is not a new result. For instance, Goto et al.50 reported an increase of almost two 4806



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Figure 5. Emission spectra of MorganElson colored product for (A) NAG standards solutions at concentrations (nmol/mL) of (stars) 1.2, (triangles pointing right) 2.3, (diamonds) 3.5, (triangles pointing down) 4.7, (triangles pointing up) 6.0, (circles) 9.2, and (boxes) 10.0; and (C) BT-HAase solutions concentrations 10 4 mg mL1 (stars) 1.2, (triangles pointing right) 2.6, (diamonds) 4.8, (triangles pointing down) 7.1, (triangles pointing up) 12.0, (circles) 18.4, and (boxes) 25.0. Calibration curves for HAase activity determination: (B) NAG and (D).BT-HAase aqueous solutions.

Table 4. Enzymatic Activity of BT-HAase in Homogeneous and Heterogeneous Media AE medium ((μmol/mg)/min)

activity

heterogeneous

0.81 ( 0.04

homogeneous heterogeneous, after 12 h

0.039 ( 0.002 0.37 ( 0.07

times in EA for catalase immobilized onto DPPG films. The explanation pointed out by the authors is based on the different factors that influence the activity of some enzymes when immobilized in nanostructured matrixes, such as orientation of the secondary structure, entrapment of the enzyme into the matrix, and accessibility of the substrate to the catalytic site. Other possible reasons that should be considered for this increased activity could be changes in reaction mechanism, changes in substrate conformation, or changes in the enzyme conformation promoted by the substrate,51 when the reaction takes place with the immobilized enzyme. In our study the enzyme is immobilized only on the top layer; thus, it is not entrapped in internal layers of the film, and one should not be aware of diffusion of the substrate within the film, and neither about the accessibility of the substrate for the film.52,53 Moreover, the estimated phospholipid:enzyme ratio of 135:1 should prevent enzyme aggregation54 and most likely

making available a larger number of active sites to form complexes with the substrate HA, which is a high molecular weight polysaccharide. In the case of BT-HAase, Belem-Goncalves et al.12 proposed that hydrophobic regions are located in the opposite side of the catalytic site region. More recently, the same group14 suggested that the interaction of the protein with a monolayer formed with a synthetic glycolipid (cholesteryl-triethoxy-N-acetylglucosamine) was very strong, suggesting the attachment of this enzyme onto the lipid film occurs by mimicking the enzymesubstrate interaction, or in other words between the hydrophilic or charged groups of HA and BT-HAase. However, no enzymatic activity assays were performed. In our case, on the basis of results of induction times for the adsorption at the interface and very high surface pressure value of coincidence between surface elasticities for pure DPPC monolayer and DPPCBT-HAase mixed monolayer, we suggest that the BTHAase hydrophobic regions are the main ones responsible for the insertion of the protein into DPPC Langmuir monolayer and LB film. In the last one, part of the hydrophobic moiety of the enzyme can also interact with the DHP matrix. As from the high EA observed for the immobilized BT-HAase, the adsorption into the film probably resulted in a preferential orientation of the enzyme, exposing its catalytic site as the model proposed in Figure 6. Moreover, most likely at our experimental conditions, the enzyme denaturation at the liquid interface was circumvented 4807



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DPPC matrix (1:∼135DPPC). Together these features can promote a higher accessibility of the substrate, HA, to the enzyme site. Therefore, the immobilization of BT-HAase in LangmuirBlodgett films arises as a very useful methodology for the fabrication of fluorimetric enzymatic biosensors based on HAase.

’ AUTHOR INFORMATION Corresponding Author

*Tel./Fax: þ55-16-3602-4838. E-mail: medzaniquelli@ffclrp. usp.br. Present Addresses †

Instituto de Ciência e Tecnologia do Mucuri, Universidade Federal dos Vales Jequitinhonha e Mucuri, Campus Avanc- ado do Mucuri, CEP 39801-000 Teofilo Otoni, Minas Gerais, Brazil. ‡ Instituto de Física de S~ao Carlos, Universidade de S~ao Paulo, Caixa Postal 369, 13560-970 S~ao Carlos, S~ao Paulo, Brazil.

’ ACKNOWLEDGMENT We thank Mr. P. C. de Sousa Filho, a Ph.D. fellow in Prof. O. A. Serra’s laboratory for his help with the spectrofluorimeter. The authors are grateful to FAPESP for the financial support. D.S.M. thanks CAPES for the Ph.D. fellowship; T.M.N. thanks FAPESP for the postdoctoral fellowship. M.E.D.Z. is a CNPq researcher. Figure 6. Schematic DPPCBT-HAase LB film model. The squares represent the phosphate hydrophilic groups of DHP in the Y-type threelayers LB film. The circles represent the phosphatidylcholine hydrophilic groups of DPPC and the narrow rectangles represents the hydrophobic lipid chains. The enzyme is buried in the lipidic film and its structure is stylized, based on data from reference 32 (1FCU-Protein Data Bank accession code). The sizes are not in proportion, and the densification of lipid molecules aims to give an idea of the 1:135 enzyme:lipid molecular ratio.

by protein injection under the lipidic monolayer, as compared to another possibility of spreading the phospholipid monolayer over an already adsorbed protein at the airliquid interface.43

’ CONCLUSIONS We have successfully assembled BT-HAase in a phospholipid LB film by choosing experimental conditions from the study of BT-HAase at a bare airliquid interface, followed by analyzing the features of the πA curves for pure DPPC and mixed DPPCBT-HAase Langmuir monolayers. We suppose BTHAase is immobilized mainly by means of dispersive forces driven by hydrophobic effect and partially by hydrogen bonding and dipole interactions between polar head groups of DPPC molecules and BT-HAase, since both are practically neutral at the pH of the work (pH 5.3). The relatively low induction times for BT-HAase at the bulk concentration range of 530 μg mL1, the coincidence of πA curves for pure DPPC and mixed monolayer only for surface pressures higher than 45 mN m1, and the high stability of the proteolipid film upon washing are results that support our hypothesis. Moreover the higher enzymatic activity exhibited by the immobilized BT-HAase, as compared with the one measured in homogeneous medium, is being attributed to a preferential orientation of the enzyme in the LB film, as well as the presence of well-distributed enzyme molecules within the

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