Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

Abstracts

This work presents equilibrium and dynamic aspects for the adsorption at the air/liquid interface of two rat osseous plate alkaline phosphatase forms: DSAP, solubilized by a surfactant, C12E9, and containing a glycosylphosphatidylinositol (GPI) anchor; and PLSAP, resulting from phospholipase-C cleavage of the hydrophobic portion of the GPI anchor. Dynamic surface tension, gammadyn, and surface elasticity modulus, epsilon, were determined for PLSAP, DSAP and pure C12E9 solutions using harmonic oscillation and axisymmetric drop shape analysis Adsorption kinetics studies revealed that DSAP adsorbs thirty times faster than PLSAP, presenting a minimum in the curve. For DSAP/ C12E9 mixed system, e increases with concentration and a maximum appears at the critical aggregation concentration (CAC). For PLSAP, a continuous decreasing with concentration for gammadyn and epsilon was observed. For pure C12E9 solution, the elasticity modulus increases with concentration and epsilon values are higher when compared to the mixed system. A model based on the influence of the GPI anchor is proposed.

dynamic surface tension; adsorption kinetics; dilatational surface elasticity; alkaline phosphatase; axisymmetric drop shape analysis

Este trabalho apresenta aspectos de equilíbrio e dinâmicos da adsorção na interface ar/líquido de duas formas de fosfatase alcalina de placa óssea de ratos: DSAP, solubilizada com tensoativo não-iônico (C12E9), contendo uma âncora de glicosilfosfatidilinositol (GPI), e PLSAP, com a porção hidrofóbica da âncora clivada por fosfolipase-C. A tensão superficial dinâmica, gamadyn, e o módulo de elasticidade superficial dilatacional, épsilon, foram determinados para soluções de PLSAP, DSAP e C12E9 pelo método de oscilação harmônica e análise do formato da gota eixo-simétrica. Cinéticas de adsorção revelaram que DSAP adsorve trinta vezes mais rapidamente que PLSAP, apresentando um mínimo, e, para PLSAP, a tensão superficial cai continuamente. Para o sistema DSAP/C12E9, épsilon atinge um máximo na concentração crítica de agregação (CAC), mas para PLSAP, épsilon diminui continuamente com a concentração. Soluções de C12E9 apresentam épsilonmais elevados, decrescentes com a concentração. Um modelo, baseado na influência da âncora GPI, é proposto para explicar os resultados obtidos.

ARTICLE

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

Luciano Caseli; Douglas C. Masui; Rosa Prazeres M. Furriel; Francisco Assis Leone; Maria Elisabete D. Zaniquelli^*

Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto-SP, Brazil

ABSTRACT

This work presents equilibrium and dynamic aspects for the adsorption at the air/liquid interface of two rat osseous plate alkaline phosphatase forms: DSAP, solubilized by a surfactant, C₁₂E₉, and containing a glycosylphosphatidylinositol (GPI) anchor; and PLSAP, resulting from phospholipase-C cleavage of the hydrophobic portion of the GPI anchor. Dynamic surface tension, g_dyn, and surface elasticity modulus, e, were determined for PLSAP, DSAP and pure C₁₂E₉ solutions using harmonic oscillation and axisymmetric drop shape analysis Adsorption kinetics studies revealed that DSAP adsorbs thirty times faster than PLSAP, presenting a minimum in the curve. For DSAP/ C₁₂E₉ mixed system, e increases with concentration and a maximum appears at the critical aggregation concentration (CAC). For PLSAP, a continuous decreasing with concentration for g_dyn and e was observed. For pure C₁₂E₉ solution_, the elasticity modulus increases with concentration and e values are higher when compared to the mixed system. A model based on the influence of the GPI anchor is proposed.

Keywords: dynamic surface tension, adsorption kinetics, dilatational surface elasticity, alkaline phosphatase, axisymmetric drop shape analysis

RESUMO

Este trabalho apresenta aspectos de equilíbrio e dinâmicos da adsorção na interface ar/líquido de duas formas de fosfatase alcalina de placa óssea de ratos: DSAP, solubilizada com tensoativo não-iônico (C₁₂E₉), contendo uma âncora de glicosilfosfatidilinositol (GPI), e PLSAP, com a porção hidrofóbica da âncora clivada por fosfolipase-C. A tensão superficial dinâmica, g_dyn, e o módulo de elasticidade superficial dilatacional, e, foram determinados para soluções de PLSAP, DSAP e C₁₂E₉ pelo método de oscilação harmônica e análise do formato da gota eixo-simétrica. Cinéticas de adsorção revelaram que DSAP adsorve trinta vezes mais rapidamente que PLSAP, apresentando um mínimo, e, para PLSAP, a tensão superficial cai continuamente. Para o sistema DSAP/C₁₂E₉, e atinge um máximo na concentração crítica de agregação (CAC), mas para PLSAP, e diminui continuamente com a concentração. Soluções de C₁₂E₉ apresentam e mais elevados, decrescentes com a concentração. Um modelo, baseado na influência da âncora GPI, é proposto para explicar os resultados obtidos.

Introduction

Surface elasticity is associated with the ability of a system to establish a new surface tension value due to a timely area variation. Considering that it is a non-equilibrium phenomenon, changes in surface tension occur either as a consequence of a sudden compression/expansion of the interface, or due to a local concentration variation. Since elasticity values are associated with diffusion processes, adsorption/desorption, as well as the rearrangement of molecules at the interface, they depend on the time scale (or frequency) in which the perturbation is applied. In addition, these processes are influenced by molecular weight, shape and molecular interactions of the species present at the interface.

Surface elasticity is directly related to the stability of emulsions and foam films,^1,2 as well as certain bio-processes occurring in cell membranes, such as, oxygen exchange in the membranes of the lung alveolae.^3,4 Moreover, some enzymatic processes occurring at the membrane level depend on the lipid/protein film surface packing and elasticity.

It has been recently demonstrated in our laboratory that there is a correlation between the equilibrium surface compressibility of a planar mixed lipid/enzyme monolayer and the catalytic activity of a glicosylphosphatidil-inositol (GPI) anchored enzyme,⁵ with the enzyme reaction occurring more efficiently for a characteristic lipid/enzyme surface packing. Such packing is associated with a surface compressibility that most likely optimizes the access of the substrate, which is dissolved in the bulk subphase, to the enzyme active site.

We have studied the p-nitrophenylphosphate hydrolysis by a rat osseous plate alkaline phosphatase. This membrane enzyme (Figure 1) can be solubilized either by non-ionic surfactants⁶ or by a glycosyl-phosphatidylinositol-specific phospholipase-C.⁷ The detergent solubilized form (DSAP) is a dimer of two apparently identical subunits of Mr 65 kDa each, and includes the intact GPI anchor.⁷ In spite of the absence of the diacylglycerol moiety,⁸ the phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase (PLSAP) shows quite close structural and catalytic properties in homogeneous medium to those of DSAP.⁹ However, its heterogeneous catalysis differs significantly from that of DSAP.¹⁰ These two enzyme forms provide a very interesting possibility for comparative studies at air/water interfaces, in order to verify the effect of the hydrophobic anchor on the surface properties.

In this work we show a comparative study of the equilibrium and dynamic properties at air/liquid interfaces of DSAP and PLSAP, employing the harmonic drop oscillation method associated to the axisymmetric drop shape analysis. To our knowledge, this is the first work reporting the effect of the GPI-anchor on the GPI-protein film surface elasticity.

Background

Some years ago, the devices employed to measure film elasticity used to be completely developed at university's or company's laboratories. Contributions were made by several research groups,^11-17 using different experimental set-ups. Among the developed techniques, the harmonic drop oscillation associated with axisymmetric drop shape analysis (ADSA) is currently commercially available.^18-20

The surface dilatational elasticity, also called surface elasticity or surface elasticity modulus, e, is defined as:

where dg accounts for the infinitesimal surface tension gradients upon a relative area (A) variation. The equilibrium value is the Young modulus and is identified as the Gibbs elasticity, also called compressibility modulus (C_s^-1) for Langmuir monolayers.

Under dynamic conditions, there are films ranging from perfectly viscous to perfectly elastic, with the latter occurring only when no relaxation process takes place in the timescale of the experiment. This fact means that there is no phase difference (described by a phase angle, q) between the perturbation (deformation) and the response of the system (change in surface tension). On the other hand, phase differences are characteristic of the interface viscoelastic behavior. This feature is accounted by introducing a complex modulus:

The imaginary part of this quantity accounts for the energy-dissipation process and is related to surface dilatational viscosity (h_s):

in which, the angular frequency, w= 2pf, is established when a sinusoidal disturbance with a frequency f is imposed to the interface.

Both the change in area and the variations in surface tension are calculated by fitting the drop shape to the Young-Laplace equation:

where DP is the difference of pressure across the interface, r_d – r_l are the densities of the denser and the lighter phases, respectively, g is the gravity acceleration, h is the height of the liquid column at the drop and R₁, R₂ the two main drop curvature radius.

Experimental

Materials

All solutions were prepared using dust free Milli-Q^® water (surface tension of 72.8 mN m^-1 and resistivity of 18.2 MW cm). Tris buffer and polyoxyethylene-9-lauryl ether (polidocanol, C₁₂E₉) were purchased from Sigma Chemical Co; purified phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis was purchased from Oxford Glyco Sciences Inc. (England). All other reagents were of the highest purity commercially available.

Preparation of detergent-solubilized alkaline phosphatase

DSAP was prepared from alkaline phosphatase-rich rat osseous plate membranes.²¹ Samples of membrane-bound alkaline phosphatase (0.2 mg mL^-1) were solubilized with 1% C₁₂E₉, (final concentration) for 2h, at 25 ºC, under constant stirring. After centrifugation at 30,000 g for 2 h, DSAP was concentrated on an YM-5 Amicon filter and dialyzed overnight against 5 mmol L^-1 Tris.HCl buffer, pH 7.5, containing 2 mmol L^-1 MgCl₂, 150 mmol L^-1 NaCl and 0.01% C₁₂E₉. Finally, DSAP was purified on a Sephacryl S-300 column (130x1.7cm) equilibrated and eluted in the same buffer. The solubilized enzyme was maintained in crushed ice for a period no longer than a month, without appreciable loss of activity. It should be emphasized that solubilization does not remove the GPI anchor.

Preparation of enzymatically-solubilized alkaline phosphatase

The phospholipase C-released rat osseous plate alkaline phosphatase (PLSAP) was solubilized from alkaline phosphatase-rich rat osseous plate membranes using PIPLC from B. thuringiensis.⁷ Briefly, aliquots (2 mg mL^-1) of membrane-bound alkaline phosphatase were incubated with 0.1 U PIPLC for 1 h, at 37 ºC. After a centrifugation at 100,000xg for 1 h, at 4 ºC, the supernatant was carefully removed and PLSAP was purified on a Phenyl-Sepharose CL-4B column (1x10 cm). The active fractions were pooled and dialyzed overnight, at 4 ºC, against 5 mmol L^-1 Tris.HCl buffer, pH 7.5, containing 2 mmol L^-1 MgCl₂. Samples of 0.1 mL were rapidly frozen in liquid nitrogen and stored at –20 ºC for a period no longer than a month without appreciable loss of activity.

Surface tension and surface elasticity measurements

Surface tension and surface elasticity were determined using an optical contact angle meter, OCA-20, with oscillating drop accessory ODG-20 from Dataphysics Instruments GmbH, Germany. A drop of liquid, formed from a syringe into a thermostated optical glass cuvette containing water in the bottom to avoid drop evaporation, is imaged using a CCD camera. A specific software that uses a suitable position for a reference line in the image field was accessed to trigger the recording of the images even before the complete drop formation. The zero time is defined after playing the movie by selecting the suitable images and surface tension was determined by firstly digitizing and analyzing the profile of the droplet. Then, the Laplace equation was fitted to the shape of the drops.

For the elasticity measurements, the images were recorded with a video camera with a minimum of 200 frames per second. At the end of the experiment, the software retrieves the images and calculates the change in area and respective changes in surface tension for each cycle. Using a Fourier transform analysis, the elasticity (e) and phase angle (q) were determined.

The drop oscillation is initiated after the surface tension reached a constant value. A piezo actuator device, connected to a function generator and located above the needle produces a sinusoidal movement of the drop in a specified frequency at the control table.

For the experiments described in this work, surface tension and elasticity measurements of aqueous solutions of C₁₂E₉, DSAP and PLSAP were carried out at 23+0.5 º C; Tris.HCl buffer, pH 7.5, was used as the heavier phase and air as the lighter phase. Frequencies between 0.1 and 1Hz were tested, and the amplitude of 0.1mm (relative area variation of 5.5%) and the frequency of 0.841Hz were arbitrarily chosen in order to compare the different systems.

Surface pressure-area isotherms

To obtain the equilibrium surface compressibilities (C_s = -1/A (¶A/¶p )_T) of DSAP monolayers at planar air/water interfaces, surface pressure (p)–area (A) compression isotherms were carried out. The buffer Tris-HCl pH 7.5 was used as subphase in a homemade trough with a volume of 40 mL and a total area of 4892 mm². Aliquots of ca. 58 µL of a 6.9 µg mL^-1 DSAP solution were spread on the top of the surface, and after 15 minutes a stable surface pressure was obtained. The surface pressure–area curves were obtained by moving the barrier at 6.73 mm² s^-1. The temperature was maintained constant at 23 + 0.5 º C.

Results and Discussion

Dynamic surface tension

PLSAP can be compared to other soluble globular proteins such as human or bovine serum albumin (HSA, BSA), ovoalbumin (OVA) or lactoglobulines, with a approximately ellipsoidal native structure. In order to investigate the mechanism of PLSAP adsorption, dynamic surface tension curves (Figure 2) were obtained using the pendant drop technique and the drop shape analysis. The adsorption kinetics at short-times is better visualized in logarithmic scale (inset of Figure 2). For a 50 µg L^-1 (3.85x10^-10 mol L^-1) concentration, the surface tension becomes stable after about 6 h. The decrease in surface tension becomes faster for more concentrated enzyme solutions, and for a concentration of 250 µg L^-1 (4.28x 10^-7 mol L^-1), the stabilization is reached in less than 3h. For higher concentrations, both the equilibrium surface tension and the equilibrium time no longer change. The induction time (time required to measure a decrease of about 0.5 mN m^-1 in surface tension – also known as lag time)²² is about 800 s. This lag time is comparable with that calculated (log t(s) = 2-3) for a model protein solution at 10^-7 mol L^-1, using a kinetic model.²³ However, they are higher than those obtained for other globular enzymes as those reported²⁴ for lysozyme and b-casein, both with low molecular masses (14.3 kDa and 24 kDa, respectively) and at higher concentrations (5-8.5 mg L^-1). For these proteins, induction times of few seconds were observed. It must be stressed that PLSAP has a very higher molecular mass (130 kDa), when compared with those proteins.

Figure 3A brings results for DSAP concentrations below and above CAC of the system (3.6x10^-6 mol L^-1 of C₁₂E₉ for 180 µg L^-1 of the enzyme). ²⁵ It is observed that for an enzyme concentration of 5 µg L^-1, (C₁₂E₉ 10^-7 mol L^-1), the system takes 15 minutes to become stable. Above the CAC, a constant surface tension of about 53 mN m^-1 is reached in less than 5 min. For this system, an induction time is not detected in the time scale of the experiment for the entire studied concentration range.

The faster adsorption kinetics of DSAP compared to PLSAP is probably due to the presence of the hydrophobic moiety of the GPI anchor in the former and absent in the latter. The anchor provides an amphiphilic character to the enzyme and is responsible for the much lower concentration necessary to decrease the surface tension (at concentrations of 5 µg L^-1, no variation in surface tension is detected for PLSAP after 12 hours of experiment). Moreover, the intact GPI anchor is also responsible for the DSAP ability to produce Langmuir monolayers²⁶ (above the CAC, the enzyme is solubilized in the buffer solution).

On the other hand, considering the absence of the hydrophobic anchor in PLSAP and, therefore, its high hydrophilic character, it is very likely that, during its air/water interface adsorption, a certain denaturation takes place at the interface due to the exposition of the hydrophobic groups.^27-31

Figure 3B shows the kinetics for DSAP adsorption in short times (less than 30s). For the highest DSAP concentration (3200 µg L^-1), the surface tension falls down continuously, reaching 52 mN m^-1 in 10 sec. For the lowest DSAP concentration studied (5 µg L^-1), the surface tension initially falls down to 63 mN m^-1, increases smoothly to 72 mN m^-1 (roughly zero surface pressure) and, after that, decreases slowly, a constant value being reached after roughly 15 min (Figure 3A). This effect is reproducible and it is observed for DSAP concentrations as high as 125 µg L^-1, but not observed for any concentration of PLSAP (Figure 2). As this process occurs only at low enzyme concentrations, there is a high area per molecule available, so it is likely that protein molecules undergo more rearrangements than it would be possible with the dense packing at the interface associated with a high enzyme concentration. Thus, this behavior may be ascribed to a three-step adsorption mechanism: (i) lateral diffusion of the enzyme at the incipient area available just before the drop formation or present in the sub-layer region, and rapid adsorption provided by the hydrophobic moiety of the GPI anchor; (ii) re-orientation of the GPI molecules at the air/water interface to a perpendicular plane relative to the interface, probably by the rearrangement of the macromolecules at the interface and (iii) adsorption of the remaining molecules present in the bulk solution.

The influence of C₁₂E₉ on the DSAP mixed system can also be verified by the dynamic surface tension for the pure non-ionic surfactant, as depicted in Figures 4A and ^B. The concentration range corresponds to the surfactant concentrations in the mixed system. In all cases, the equilibrium surface tension is roughly 10 units lower for the pure surfactant when compared to the mixed system. The time to reach a constant surface tension is longer than that required for the mixed system, in which the insoluble-in-water enzyme is present. For a concentration of about 1.0x10^-7 mol L^-1, approximately two and a half hours are necessary for the surface tension to reach a stable value, similarly as reported by Ravera et al.³² for C₁₀E₅. However, when C₁₂E₉ concentration is increased to or higher than 3.26 x 10^-5 mol L^-1, only a few minutes (less than 20min) are necessary to reach the equilibrium surface tension. ^{Figure 4B} shows the kinetics of adsorption of C₁₂E₉ at short times. An induction time is observed for the lowest concentration. However, differently from the results obtained for DSAP, no minimum is seen in the curve for pure C₁₂E₉ in a concentration equivalent to that in the mixed system. This behavior suggests that the effect observed for the mixed system is, in fact, mainly due to the protein component of the mixture.

Elasticity measurements

Figure 5 shows an example of surface tension variation in response to the deformation of the interfacial area. From this example, the phase angle is practically zero, which means that the interface has a perfect elastic behavior. A deviation from zero corresponds to viscoelastic effects,¹³ which are in fact observed for some of the analyzed frequencies. However, in all cases, the elastic component is dominant.

As the elasticity modulus (e) depends on the frequency, this parameter was changed from 0.01 to 1.0 Hz. This frequency range corresponds to relaxation times characteristic for different proteins^33,34 and is associated to rearrangement processes. As the obtained results show the same tendency, the frequency of 0.841 Hz and relative area variation of 5.5% (corresponding to the mechanical amplitude of 0.1 mm of the piezo system) were arbitrarily chosen to compare the data for the two proteins and for the non-ionic surfactant. This amplitude is small enough to prevent the detachment of the drop from the syringe tip during the compression/expansion process and also does not impart problematic diffusion/convection effects.

The dependence of the dilatational elastic moduli (e) on the concentration for DSAP is shown in Figure 6. Elasticity for C₁₂E₉ is shown in the same plot for comparison. For C₁₂E_9, the elasticity decreases continuously with the concentration. In previous studies using other polyoxyethylene alkyl ether surfactants and similar frequencies,³⁵ a small decrease in the elasticity modulus has been observed. However, the concentration range used in that work (5x10^-12 – 10^-10 mol L^-1) was lower than that used in this work (10^-7-10^-4 mol L^-1). In addition, for C₁₂E₄, Stuberauch and Miller³⁶ reported a decrease of the elasticity modulus with increasing surfactant concentrations (in the range of 10^-5-10^-4 mol L^-1) at a frequency lower than 1.00Hz.

On the other hand, in the case of DSAP the elasticity reaches a maximum at the CAC. According to some authors,³⁷ there is a competitive adsorption between the constituent species of mixed soluble protein/non-ionic surfactant systems. DSAP constitutes, however, a peculiar system due to the presence of the GPI anchor, and its solubility in water, as well as its adsorption, depends on the non-ionic surfactant concentration. From Figure 6, it may be observed that the CAC represents a limit to the elasticity. For higher concentrations, there is a reduction in e, meaning that the solubilization of the GPI-anchored protein makes the air/water interface more resistant to a gradient of surface tension due to diffusive effects. Moreover, between C₁₂E₉ concentrations of 3.6x10^-6 and 7.1x10^-5 mol L^-1, CAC and CMC respectively, the elasticity values for the DSAP/ C₁₂E₉ system and pure C₁₂E₉ are close. In contrast, for concentrations higher than the CAC, the elasticity modulus for pure C₁₂E₉ continuously decreases, whereas the mixed system maintains a constant elasticity. This fact indicates that the interaction of the DSAP anchor with C₁₂E₉ can limit the diffusion of the non-ionic surfactant (or even of the enzyme) to the interface.

In order to compare the dynamic dilatational elasticity measured at the drop surface with the equilibrium compressional modulus at the planar interface, surface pressure-area (p-A) compression isotherms were obtained (Figure 7). At selected pressures, the compressional moduli, taken as -dp/dlnA, are displayed on the graph. Table 1 compares the dynamic surface elasticity and the equilibrium surface compressional modulus. The values obtained at the same pressures do not differ more than 11% from each other, being the highest difference observed at 19.9 mN m^-1, close to the collapse pressure (20.2 mN m^-1). For surface pressures above 20 mN m^-1, the values cannot be compared because the phenomena occurring in the two systems are different. For the equilibrium regime (Langmuir trough) the monolayer attained the "collapse", and due to the high volume of the subphase (around 40 mL), the concentration of the enzyme/C₁₂E₉ system is still below the CAC and, therefore, the enzyme cannot be solubilized. At the collapse, there is a rearrangement at the air/water interface, with the possibility of multilayer formation. On the other hand, in the drop, the surface tension of 20 mN m^-1 is attained with increasing concentrations of the enzyme/C₁₂E₉ system in such a way that the CAC is attained, and molecules can be displaced to the bulk solution (subphase).

The elasticity for the globular soluble PLSAP (Figure 8) was also studied. It decreases continuously with concen-tration, showing a more pronounced decay at 10^-2 µg L^-1. The elasticity values (between 10 and 14.5 mN m^-1 for the frequency studied) are lower when compared with those reported for other hydrophilic and globular proteins. The elasticity moduli for air/water interfaces for solutions of standard proteins with lower molecular weights, like BSA, HSA, b-casein and b-lactoglobulin, were measured by many authors, using various techniques.^31,38-41 For instance, b-lactoglobulin (M_r 18.4 kDa) shows an average elasticity of 26.04 mN m^-1 for a frequency of 0.0125Hz, at a concentration around 1x10^-7mol L^-1.³³ Nevertheless, Benjamins et al.³⁸ have already pointed out that the interfacial pressure rather than the bulk concentration is the most relevant parameter to compare the moduli. Thus, the value of 17.3 mN m^-1 obtained for BSA, measured at a surface pressure of 56.2 mN m^-1, should be compared to the elasticity obtained for other globular proteins at the same surface pressure. Table 2 summarizes a comparison between our results and other elasticity values obtained from the literature. Compressibility for in-planar surface PLSAP monolayers was not obtained due to the high solubility of the enzyme, and the use of high ionic strength, which could prevent the its solubilization, was not employed due to the likely denaturation of the enzyme.

Furthermore, PLSAP exhibits lower values of e, if compared to DSAP (see Figures 6 and 8). This fact can be attributed to the higher DSAP surface activity due to the GPI anchor. Since the surface dilatational elasticity indicates the ability of the interface to restore the original surfactant surface density providing a surface tension gradient, the GPI-anchored enzyme is able to resist to changes in area much more efficiently than the globular soluble enzyme without the anchor. Thus, the GPI-anchored enzyme DSAP can also be associated to a better ability to stabilize films.¹⁴ Moreover, the elasticity for both proteins must be associated to a reversible movement of the protein segments,⁴² which could also be a relevant factor for enzymatic processes. However, as the DSAP form contains a hydrophobic anchor, it is strongly likely that its major elasticity effects are related to the capability of the anchor to be adsorbed on air/water interfaces. Since few enzyme molecules are present in the bulk solution, the Gibbs mechanism for surface pressure regeneration can be neglected, which provides the higher enzyme ability in resisting to an area gradient, followed by a surface tension gradient.

The dilatational effects for both enzyme forms may be also associated with different conformation and orientation changes at the air/water interface. Alkaline phosphatase has an ellipsoidal shape,⁴³ and, according to a previous work,¹⁰ its orientation at the interface (major axis parallel or perpendicular to the interface) is strongly influenced by the presence or not of the hydrophobic anchor. Therefore, the higher or lower capability of the enzyme molecules to pack at the interface, as well as the protein solvation^20,44-45 may lead to the different velocities of adjustments of the steady state. In this sense, to rebuild the surface structure with a high mechanical stability, the conformational and orientation changes must not be highly restricted. In our case, the presence of a phospholipid (hydrophobic anchor) covalently bound to the polypeptidic moiety of the protein gives the GPI-anchored alkaline phosphatase extremely peculiar surface properties, with the GPI-anchor driving the processes of adsorption, orientation,^10,25,46-48 and packing⁹ and, as stated by the present work, also influencing the surface elasticity properties. To the best of our knowledge, this is the first report concerning the elasticity properties of a GPI-anchored protein.

Conclusions

We have demonstrated that the enzyme form lacking a hydrophobic anchor (PLSAP) exhibits surface activity due to the adsorption of the polypeptide moiety of the enzyme at the air/water interface. As expected, the hydrophobic anchor-containing form (DSAP) has a higher surface activity due to the presence of the hydrophobic anchor. The elasticity of alkaline phosphatase at a certain frequency and amplitude depends on the following factors: presence of the GPI anchor, concentration, surface pressure, solubility induced by non-ionic surfactant and surface packing. The anchor-containing form (DSAP) has an elasticity modulus higher than the lacking-anchor form (PLSAP) at the same surface pressure. In the studied frequency, the elasticity modulus is associated to the CAC of the surfactant/enzyme system. Since PLSAP has a high mobility due to its hydrophilic character and low concentration, it displays elasticity values that decrease with concentration. This work will certainly help the understanding of the rheological behavior of enzyme/phospholipid interfaces and also state the differences in the dilatational elasticity of the liquid interface in the presence of enzymes.

Acknowledgments

The authors thank FAPESP for financial support and are grateful to Dr. T.Holz and Dr. C. M.C. P. Manso for the careful reading of the manuscript, to Dr. I. Borin for adaptation of Figure 1. L. Caseli thanks FAPESP for his Ph.D. fellowship. M.E.D.Zaniquelli and F.A.Leone are CNPq research fellows.

Received: February 14, 2005

Published on the web: July 27, 2005

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  09 Nov 2005
• Date of issue
  Oct 2005

History

• Accepted
  27 July 2005
• Received
  14 Feb 2005