Adsorption of detergent-solubilized and phospholipase C-solubilized alkaline phosphatase at air/liquid interfaces

Adsorption of detergent-solubilized and phospholipase C-solubilized alkaline phosphatase at air/liquid interfaces

Luciano Caseli, Maria Elisabete Darbello Zaniquelli *,Rosa Prazeres Melo Furriel, Francisco Assis Leone

Departamento de Quımica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, 14040-901 Ribeirao

Preto, SP, Brazil

Accepted 10 April 2003

Abstract

To investigate the influence of a hydrophobic anchor on protein adsorption, equilibrium and dynamic aspects of the

adsorption of two different solubilized forms of rat osseous plate alkaline phosphatase on Langmuir monolayers of

dimyristoylphosphatidic acid (DMPA) were studied. Surface pressure and surface potential measurements at air/liquid

interfaces were carried out using the detergent-solubilized form (DSAP) of alkaline phosphatase, which holds a

glycosylphosphatidylinositol (GPI) hydrophobic anchor, and the glycosylphosphatidylinositol-specific phospholipase

C-solubilized form (PLSAP), lacking the GPI anchor. Similar surface transitions observed for both DMPA and

DMPA/PLSAP mixed monolayers indicate that the presence of PLSAP does not promote significant changes in surface

packing of the DMPA monolayer. However, PLSAP interacts with the polar portion of the phospholipid even at high

lateral compression. The presence of the GPI anchor increases the adsorption of DSAP at a plain air/liquid interface

and also enables the penetration of the protein into the DMPA monolayers. The penetration is dependent on both time

and surface pressure. Up to 20 mN/m, the surface pressure increases smoothly indicating a diffusion followed by an

adsorption process. Above 20 mN/m, after a fast increase, the surface pressure slowly decays to equilibrium values quite

close to the initial surface pressures. The results indicate that the molecular packing of the lipid layer drives the enzyme

adsorption to the interface either through the GPI anchor or by the polypeptide moiety.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Alkaline phosphatase; Dimyristoylphosphatidic acid; Langmuir monolayers; Adsorption kinetics

1. Introduction

Living cells, cellular membranes and membrane

models are often used to study biosensors, bior-

eactors and also the biophysical and biochemical

phenomena involved in the interaction between

molecules and cellular membranes [1,2]. Lipid

bilayers, vesicles and/or monolayers are the most

* Corresponding author. Tel.: �/55-16-602-4373; fax: �/55-

16-633-8151.

E-mail address: [email protected] (M.E.D.

Zaniquelli).

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0927-7765/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

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commonly used systems and each one has parti-cular advantages depending on the aspect or

application of interest [3�/5]. The Langmuir tech-

nique is based on the property of amphiphilic

molecules to form a compact monomolecular film

when spread at the air/water interface and subse-

quently subjected to compression [6�/9]. Studies

using this monolayer system permit improve

understanding of enzyme�/phospholipid matrixinteractions and characterization of the factors

that can lead to higher enzymatic activity and

stability [10,11]. In addition, surface pressure and

surface potential isotherm studies can provide

macroscopic information on how the protein�/lipid

interactions occur, and the dynamics of such

interactions [3,12]. Due to their ubiquitous dis-

tribution and the relative ease of preparation andpurification, alkaline phosphatases are suitable

enzymes to be used for biosensor and biotechno-

logical applications [13�/16]. Alkaline phosphatase

from rat osseous plate is a multifunctional phos-

phomonohydrolase tightly associated to the cellu-

lar membrane through a glycosylphosphatidyl-

inositol (GPI) anchor [17] and is considered to be

involved in the biomineralization process [18]. Thisenzyme has been successfully solubilized using

either polyoxyethylene-9-lauryl ether [19] or gly-

cosylphosphatidylinositol-specific phospholipase

C [20], and purified to homogeneity. The detergent

solubilized form (DSAP) is a dimer of two

apparently identical subunits of about 65 kDa

that include the intact GPI anchor [20]. The

phosphatidylinositol-specific phospholipase C-so-lubilized alkaline phosphatase (PLSAP) shows

quite close structural and catalytic properties in

homogeneous medium to those of DSAP [17] but

the diacylglycerol moiety [21] is absent. The lack of

the diacylglycerol moiety, presents a very interest-

ing possibility for comparative adsorption studies

on Langmuir monolayers of different forms of a

given enzyme.Alkaline phosphatases from several sources

have been intensively studied at air/buffer inter-

faces [15,22�/26]. Polarized infrared light reflec-

tance studies of the membrane-anchored enzyme

form in the presence of two different phospholi-

pids have shown that the enzyme orientation at the

air/liquid interface is independent of the presence

of the phospholipid [25]. High ionic strength hasalso been used to induce the adsorption of a

soluble form of alkaline phosphatase at the air/

liquid interface, but little attention was given to

the orientation of the enzyme [15].

In this work, equilibrium and dynamic aspects

of DSAP and PLSAP adsorption at both plain air/

buffer interface and Langmuir monolayers of

dimyristoylphosphatidic acid (DMPA) were stu-died. To our knowledge, this is the first report

showing both dynamic and equilibrium aspects of

the adsorption of two solubilized forms of the

same enzyme to lipid Langmuir monolayers. In

addition to an improvement in the understanding

of the relative contributions of the polypeptide

moiety and of the hydrophobic anchor to the

interaction of the enzyme with the phospholipidmatrix, our results may clarify the factors that

affect the transfer of these proteins to solid

substrates. Further, possible differences in the

enzymatic activity observed for reactions occur-

ring at air/liquid interfaces will be better explained

after a complete characterization of enzyme ad-

sorption.

2. Materials and methods

All solutions were prepared using dust free

Millipore MilliQ ultrapure water. Polyoxyethy-

lene-9-lauryl ether (C12(EO)9), DMPA, 2-amino-

2-methyl-propan-1-ol (AMPOL) and p -nitrophe-

nylphosphate (PNPP) were purchased from SigmaChemical Co. Chloroform and methanol were

from Merck. Am241 was from Amersham (UK).

All other reagents were of the highest purity

commercially available. Gold-covered quartz crys-

tals were purchased from International Crystal

Manufacturers (USA). Purified phosphatidylino-

sitol-specific phospholipase C (PIPLC) from B.

thuringiensis was purchased from Oxford Univer-sity (UK).

2.1. Preparation of enzymatically-solubilized

alkaline phosphatase

1.0 ml aliquots (2 mg/ml) of rat osseous plate

membrane-bound alkaline phosphatase [27] in 50

L. Caseli et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 273�/282274

mmol/l Tris�/HCl buffer, pH 7.25, were incubatedwith 0.1 U PLSAP, for 1 h and 37 8C, under

constant rotary shaking [20]. After a centrifuga-

tion at 100 000�/g for 1 h and 4 8C, the super-

natant was carefully removed and the

phospholipase C-released rat osseous plate alka-

line phosphatase (PLSAP) was purified as follows.

The ionic content of the supernatant was altered to

give final concentrations of 1 mM ZnCl2, 2 mmol/lMgCl2 and 2.7 M NaCl, with gentle stirring at

4 8C, adjusted to pH 7.5 and applied to a Phenyl-

Sepharose CL-4B column (1�/10 cm) previously

equilibrated with 5 mmol/l Tris�/HCl buffer, pH

7.5, containing 1 mM ZnCl2, 2 mmol/l MgCl2 and

2.7 M NaCl. Stepwise elution was carried out with

decreasing NaCl concentration in the buffer.

Fractions of 1.5 ml were collected at a flow rateof 18 ml/h and the active fractions were pooled

and dialyzed overnight, at 4 8C, against 5 mmol/l

Tris�/HCl buffer, pH 7.5, containing 2 mmol/l

MgCl2. Samples of 0.1 ml were rapidly frozen in

liquid nitrogen and stored at �/20 8C for a period

no longer than a month without appreciable loss

of activity.

2.2. Preparation of detergent-solubilized alkaline

phosphatase

Samples containing 0.2 mg/ml of rat osseous

plate membrane-bound alkaline phosphatase were

solubilized with 1% polidocanol (final concentra-

tion) for 2 h and 25 8C with constant stirring. After

centrifugation at 100 000�/g for 2 h, DSAP wasconcentrated on an YM-5 Amicon filter and

dialyzed overnight against 5 mmol/l Tris�/HCl

buffer, pH 7.5, containing 2 mmol/l MgCl2, 150

mmol/l NaCl and 0.01% polidocanol. Finally,

DSAP was purified on a Sephacryl S-300 column

(130�/1.7 cm) equilibrated and eluted in the same

buffer used for dialysis according to Ciancaglini et

al. [19].

2.3. Surface pressure�/area isotherms

The surface pressure�/area isotherms (p �/A iso-

therms) of DMPA were obtained at 239/1 8C, with

a homemade Langmuir trough [28] equipped with

a Cahn microbalance model C-32. DMPA mono-

layers were obtained by spreading 1 mmol/lDMPA solution (dissolved in 3:1 chloroform:-

methanol) on an air/buffer interface. Mixed mono-

layers of DMPA and the two different forms of the

enzyme were prepared by pre-spreading of DSAP

or PLSAP solutions. DMPA was spread after the

surface pressure and surface potential stabilization

(about 10 min). Only after a new stabilization of

the surface pressure, a surface compression of 0.56cm2/s was initiated. In all cases, 5 mmol/l Tris�/

HCl buffer, pH 7.5, containing 2 mmol/l MgCl2was present in the subphase and is referred in the

text as simply ‘‘subphase buffer’’. Zero of surface

pressure and surface potential were considered as

the values measured for the air/buffer interface

before DMPA and protein spreading.

2.4. Surface potential�/area isotherms

The surface potential�/area isotherms (DV �/A

isotherms) were measured by the radioactive

electrode method [29] using Am241 and a saturated

calomel reference electrode. The measurements

were performed using a 617 computer-interfaced

model Keithley electrometer confined in a Faradaycage. Changes in the surface potential and surface

pressure were simultaneously recorded during the

compression of the monolayers.

2.5. Surface tension curves

Surface tension curves of PLSAP and polidoca-

nol/DSAP mixtures were determined using a KSV701 model Sigma automatic tensiometer. Enzyme

solutions were injected into buffer solution, at

239/1 8C and the measurements were recorded 15

min after the enzyme injection. After this period

no significant changes in the surface tension were

detected. The enzyme was injected either under a

plain air/buffer interface or under a DMPA

monolayer at a surface pressure of about 6 mN/m (area per molecule of ca. 50 A2).

2.6. Adsorption kinetics of DSAP and PLSAP at

air/buffer interface

The adsorption of DSAP and PLSAP at air/

liquid interfaces was investigated at 239/1 8C, by

L. Caseli et al. / Colloids and Surfaces B: Biointerfaces 30 (2003) 273�/282 275

monitoring the surface pressure variations withtime after injecting the enzyme into the subphase

support (5 mmol/l Tris�/HCl buffer, pH 7.5,

containing 2 mmol/l MgCl2) of the monolayer.

The injection was carried out using different initial

surface pressures. The effect of enzyme concentra-

tion (1.0, 2.5 and 3.5 mg/l) on surface tension was

measured both at the plain air/buffer interface and

in the presence of the DMPA monolayer.

3. Results

3.1. Surface pressure and surface potential

isotherms for DMPA monolayers formed on sub

phases containing DSAP and PLSAP

Fig. 1 shows the effect of DSAP and PLSAP onthe surface pressure (p)�/ and surface potential

(DV )�/area isotherms of DMPA monolayers. The

marked differences observed for DMPA/DSAP

mixed monolayer isotherm are a consequence of

the highly amphiphilic character of DSAP. The

coexistence region between the liquid-expanded

(LE) and liquid-condensed (LC) states observed

for the DMPA monolayer was abolished in thepresence of DSAP, whereas for PLSAP-containing

mixtures the LE�/LC transition occurred at lower

surface pressures and with a reduced area per

molecule range (Fig. 1A). Furthermore, the mini-

mum areas observed for DMPA and DMPA/

DSAP mixtures were almost coincident. Although

the presence of PLSAP apparently does not affect

the general type of transition observed for DMPAmonolayers, the minimum area per molecule was

higher, with values of about 45 A2 for DMPA/

PLSAP mixtures, as compared with pure DMPA

monolayers, of around 41 A2. For areas per

phospholipid molecule greater than 90 A2, the

coincidence of both p �/A isotherms does not

contribute to elucidate the interaction between

PLSAP and DMPA.The surface potential�/area isotherms for

DMPA monolayers in the presence of DSAP and

PLSAP are shown in Fig. 1B. For areas of about

90 A2 per molecule, the DMPA/PLSAP monolayer

exhibits a surface potential 60 mV higher than that

observed for the pure DMPA monolayer (0 mV).

A similar difference was observed between the

maximal DV for the DMPA/PLSAP (420 mV) and

pure DMPA monolayers (350 mV). These differ-

ences in DV values could be a consequence of the

contribution of the enzyme dipole moment since

the similarity of the isotherm profiles suggests that

the polypeptide moiety does not affect signifi-

cantly the inclination of DMPA chains during the

compression. However, the plateau region ob-

Fig. 1. Surface pressure and surface potential�/area isotherms

for DMPA monolayers. (A) p �/A isotherm. (B) DV �/A

isotherm. The isotherms were recorded simultaneously at 239/

1 8C, at a compression rate of 0.56 cm2/s started only after the

stabilization of the surface pressure and 5 mmol/l Tris�/HCl

buffer, pH 7.5, containing 2 mmol/l MgCl2 present in the

subphase; (j) buffer only, (m) in the presence of 2.5 mg/l

DSAP, (') in the presence of 2.5 mg/l PLSAP.


served for PLSAP/DMPA isotherm (from 58 to 78A2 per molecule) shows a displacement relative to

that of DMPA (of about 52�/72 A2), which

suggests some interaction between DMPA and

PLSAP. For the DMPA/DSAP monolayer, the

surface potential increases monotonically and the

maximum value observed was about 210 mV,

suggesting considerable differences between this

mixed monolayer and that constituted by DMPAonly. Taken together, the differences observed in

DMPA/DSAP and DMPA monolayer profiles are

apparently associated to the contribution of the

hydrophobic anchor to the surface potential.

Further, the significantly reduced value of the

maximum surface potential of about 210 mV at

high compression of the DMPA/DSAP monolayer

suggests a lower tilt angle for the hydrophobicchains of the phospholipid, which results in a

smaller contribution to the dipole moment in the

normal direction.

3.2. Equilibrium adsorption of DSAP and PLSAP

at the air/buffer interface

For a constant trough area, the adsorption of

different DSAP concentrations at the plain air/buffer interface resulted in a maximal surface

tension of 63.7 mN/m for an enzyme concentration

of 4.4 mg/l, which corresponded to 10�7 mol/l

C12(EO)9. The maximal surface tension obtained

for C12(EO)9 buffered solution, at the same con-

centration as above, was 58.3 mN/m (Fig. 2A).

The addition of different concentrations of pure

C12(EO)9 caused a more significant effect on thesurface tension, which can be explained by the

easier packing of the surfactant in the absence of

the enzyme. Considering that the DSAP solution

has a 560:1 surfactant:enzyme mole ratio, the

surfactant�/enzyme interaction within the en-

zyme�/surfactant complex (DSAP) contributes to

the displacement of the surfactant from the inter-

face.The adsorption of DSAP at the air/buffer inter-

face was apparently affected in a different way by

the presence of the DMPA monolayer (Fig. 2B).

At an initial surface pressure of ca. 6 mN/m,

increasing concentrations of DSAP from 4.5 to

11.2 mg/l (corresponding to the surfactant concen-

tration range from 10�7 to 3.2�/10�7 mol/l)

caused a decrease from 58.0 to 53.0 mN/m in the

surface tension. A decrease of 2.2 mN/m (from

66.0 to 63.8 mN/m) in the surface pressure was

observed when pure C12(EO)9 was used over the

same concentration range. Extrapolation of the

concentrations of both DSAP and C12(EO)9 to

infinite dilution (5 mmol/l Tris�/HCl buffer, pH

7.5, containing 2 mmol/l MgCl2), resulted in a

surface pressure of 6 mN/m (not shown) upon

which DMPA monolayer reaches the LE�/LC

Fig. 2. Surface tension curves for DSAP recorded at constant

area trough; (A) buffer only, (B) in the presence of a preformed

DMPA monolayer at 6.4 mN/m, prepared on 5 mmol/l Tris�/

HCl buffer, pH 7.5, containing 2 mmol/l MgCl2 at 239/1 8C.

(j) DSAP, (m) data for C12(EO)9 shown for comparison.


coexistence region. The presence of the monolayerin this state seems to make a positive contribution

to the adsorption of DSAP and is not observed for

C12(EO)9, which is perhaps due to the single

surfactant chain in contrast to the double lipid

chain present in the anchor-containing DSAP.

Fig. 3 shows the effect of DMPA on the surface

tension for different concentrations of PLSAP.

The decrease in surface tension was more evidentin the absence of the DMPA monolayer since

PLSAP does not have the diacylglycerol moiety. In

spite of the lower effect of PLSAP on the surface

tension (from 64.8 to 60.6 mN/m) compared with

DSAP (from 61.8 to 51.9 mN/m, showed in Fig.

2B), the profiles of the surface tension curves for

both PLSAP and DSAP in the presence of DMPA

are characteristic of surface saturation associatedwith some bulk aggregation.

3.3. Kinetics of DSAP and PLSAP adsorption at

the air/liquid interface

Fig. 4 shows the kinetics of DSAP adsorption at

the air/buffer interface and in the presence of

DMPA monolayers. In the absence of DMPA,

maximal surface pressure of about 3.5 mN/m was

observed after protein addition to the bulk solu-

tion (Fig. 4A), independent of protein concentra-

tion (1�/3.5 mg/l). The increasing protein

concentration from 1 to 3.5 mg/l resulted in

decreased time (600 to about 200 s) to attain

equilibrium values, which is consistent with a

diffusion-controlled process. In contrast, in the

presence of DMPA (Fig. 4B), the injection of a

fixed concentration of DSAP (3.5 mg/l) at different

Fig. 3. Surface tension curves for PLSAP recorded at constant

area trough. The isotherms were recorded at 239/1 8C; (m) in

the presence of a preformed DMPA monolayer at 6.4 mN/m

prepared on 5 mmol/l Tris�/HCl buffer, pH 7.5, containing 2

mmol/l MgCl2; (j) buffer only.

Fig. 4. Effect of DMPA on the adsorption kinetics of DSAP.

The surface pressure was monitored at 239/1 8C, immediately

after the injection of the enzyme under the air/liquid interface;

(A) buffer only, (j) 1.0 mg/l DSAP, (m) 2.5 mg/l DSAP, (') 3.5

mg/l DSAP; (B) 3.5 mg/l DSAP in the presence of a preformed

DMPA monolayer at initial pressure of 0 mN/m (j); 4.5 mN/m

(m); 10 mN/m (%); 15 mN/m ("); 20 mN/m (�/); 25 mN/m

(�/) and 30 mN/m ('). Inset; expanded scale plot for the first

200 s, at initial surface pressure of 0 mN/m.


initial surface pressures (pi) resulted in variabletimes to attain equilibrium values (pe). As pi

increased from 0 to 15 mN/m, the equilibrium

time was reduced from 150 to 50 s. Furthermore

for an initial surface pressure of about zero, a lag

period was observed (inset to Fig. 4B), and above

4.5 mN/m, this lag period disappeared suggesting

that DSAP was almost instantaneously adsorbed

at the air/buffer interface. For initial surfacepressures equal or higher than 20 mN/m, striking

modifications on the profiles were observed. After

an abrupt increase in the first 50 s, the surface

pressure decreased smoothly to equilibrium values

ranging from 20 to 30 mN/m, depending on the

initial surface pressure. Similar results were ob-

tained for protein concentrations of 1.0 and 2.5 mg/

l (not shown). A summary of the kinetic resultsobtained for protein concentration of about 3.5

mg/l is given in Table 1. No significant changes in

the surface pressure were observed for PLSAP

concentrations up to 10 mg/l, under the same

conditions as described above. Unfortunately,

higher PLSAP concentrations could not be used

due to the large volume of the trough employed.

4. Discussion

4.1. Role of the anchor in the adsorption of alkaline

phosphatase

The influence of the GPI hydrophobic anchor in

the adsorption of a mammalian alkaline phospha-

tase at an air/buffer interface and at Langmuirmonolayers has been studied. Our data show that

the presence of PLSAP has only a minor effect on

the surface pressure values for DMPA (see Fig. 1).

It has previously been reported that non-anchored

polypeptides interacting solely with the lipid head

groups through electrostatic interactions and with-

out penetration into the monolayer do not affect

the surface pressure [30]. However, the shift in areaper phospholipid molecule observed for the

DMPA and DMPA/PLSAP p �/A isotherms sug-

gests that the molecular packing of DMPA at high

phospholipid surface density is influenced by the

presence of PLSAP. Considering the extreme case

in which all the protein molecules remain at the

air/buffer interface, this increase in area corre-

sponds to 6600 A2 per enzyme molecule. Takinginto account the high solubility of the enzyme in

the buffer, the larger area available per protein

molecule was apparently caused by the PLSAP/

DMPA head group interaction.

The differences observed for the effect of

increasing concentrations of DSAP and PLSAP

on the surface tension curves of an air/buffer

interface and DMPA monolayers (see Figs. 2 and3) corroborate the importance of the GPI anchor

for the adsorption of the protein in the phospho-

lipid monolayers. Indeed, large changes in the

surface tension are not expected to occur for

hydrophilic macromolecules, and with DMPA

monolayers, the presence of the phospholipid at

the air/liquid interface represents a barrier to

PLSAP adsorption (see Fig. 3). The fact that thesurface pressure isotherms (Fig. 1) and the surface

tension curves (Figs. 2 and 3) are less affected by

PLSAP than DSAP confirms that the absence of

the diacylglycerol moiety in PLSAP resulted in a

lower surface activity. The presence of the hydro-

phobic anchor is apparently involved in the

packing of the phospholipid molecules (see Fig.

1), since significant changes in the isothermprofiles were observed in the presence of DSAP

and a minor displacement of the minimum area

was observed for DMPA monolayer in the pre-

sence of PLSAP. However, our data do unequi-

vocally exclude the possibility that PLSAP

undergoes partial unfolding, which exposes hydro-

phobic residues at the air/buffer interface.

Table 1

Dynamic adsorption parameters of DSAP (3.5 mg/l) injected

under DMPA Langmuir monolayers at different initial surface

pressures

Initial surface pres-

sure (pi) (mN/m)

Equilibrium surface

pressure (pe) (mN/m)

Equilibrium

time (s)

0 3.4 150

4.5 6.0 75

10 11.7 75

15 15.9 50

20 21.2 150

25 25.5 550

30 30.0 550


Polarization modulation-infrared reflectanceabsorbance spectroscopy (PM-IRRAS) analysis

of GPI-anchored alkaline phosphatase with anio-

nic and neutral phospholipid layers has shown that

the presence of the anchored enzyme induces more

pronounced disorder of the hydrophobic phos-

pholipid monolayers in the presence of an anionic

polar head phospholipid [24,26]. This suggests that

the electrostatic interaction between the chargedpolar head and the charged groups of the enzyme

drives the reorientation of the phospholipid

chains. On the other hand, the overall orientation

of the enzyme at the air/liquid interface is not

affected by the presence of the phospholipid

monolayer [26]. In light of the PM-IRRAS data

[24,26] the surface potential results presented here

for DMPA/DSAP can be interpreted as having amajor contribution of the hydrophobic tails. If the

phospholipid monolayers are more disorganized in

the presence of the enzyme, their average tilt angle

relative to the interface should decrease, providing

a lower surface potential for DMPA/DSAP com-

pared with pure DMPA. However, the interaction

between PLSAP and DMPA does not affect the

orientation of the hydrophobic chains. This can beobserved by subtracting the polypeptide moiety

contribution for the surface potential observed at

larger areas from the whole curve (Fig. 1B). As a

consequence, the resulting surface potential iso-

therm is similar to that obtained for pure DMPA.

4.2. Influence of the nonionic surfactant in the

DSAP adsorption

It is shown (Fig. 2B) that pure C12(EO)9 is not

significantly adsorbed into the DMPA monolayer,

which is indicated by the slight decrease in the

surface tension in the presence of polidocanol

concentration up to 3.2�/10�7 mol/l. The addi-

tion of C12(EO)9 into pure DMPA monolayer

subphase results in an effect similar to a compres-

sion of the interface, but the pure surfactant cannot abolish the LE�/LC transition occurring in the

range of 44�/64 A2 per molecule observed in Fig.

1A. However, enzyme�/surfactant mixtures re-

sulted in a decreased surface tension at the same

polidocanol concentration range (see Fig. 2B).

These data are reminiscent of those showed in

Fig. 1A, in which the main transition is absent inthe DSAP/DMPA p �/A isotherm.

4.3. Dynamic aspects of the adsorption

Kinetic effects can play an important role in the

adsorption of macromolecules, affecting the dy-

namic properties of the interface and revealing

possible adsorption mechanisms [31,32]. Data

from Table 1 suggest that as pi is graduallyincreased from 0 to 20 mN/m, the adsorption of

DSAP is facilitated by the fact that the phospho-

lipid chains are bringing together with their

hydrophobic chains driven to an uppermost direc-

tion related to the interface. As a result, decreasing

times to attain the equilibrium surface pressure are

observed as pi increased from 0 to 20 mN/m. For

pi]/20 mN/m, in addition to significant changes ofthe adsorption kinetics, the time to attain pe also

increases significantly. This transition surface

pressure (20 mN/m) has been named by some

authors [24] as exclusion surface pressure, and it

has been interpreted as the consequence of mono-

layer condensation, which prevents the penetration

or causes the expulsion of the enzyme from the

monolayer. On the other hand, it has beenreported that mixed phospholipid monolayers of

alkaline phosphatase compressed to a surface

pressure of about 30 mN/m, exhibit increased

signal associated with the amide content of the

enzyme than that observed at lower pi, which

suggests that a significant adsorption of the

enzyme occurs at high values of p [26], what

confirms the presence of the protein even at highpressures and also rules out the possibility of total

expelling of the protein from interface.

In the case of DSAP, assuming that the enzyme

is not expelled from the interface, the rapid

increase of p followed by the smooth decrease to

pe values should be attributed to the mechanism of

DSAP adsorption. Apparently this mechanism

could involve enzyme adsorption through theaminoacid moiety and/or through the hydropho-

bic anchor, depending on the phospholipid pack-

ing. Enzyme penetration in the DMPA monolayer

will occur in all cases, but the hydrophobic anchor

will be directed to the air/water interface. In

conclusion, up to 20 mN/m, the polypeptide


moiety orientation occurs randomly, and theenzyme apparently exposes some hydrophobic

residues at the interface. As the DMPA monolayer

becomes tightly packed, the adsorption through

the aminoacid moiety is unlikely and the adsorp-

tion occurs via penetration of the hydrophobic

anchor into the dense monolayer. This is consis-

tent with the rapid increase in the surface pressure,

since the anchor holds the polypeptide moiety.Furthermore due to the large volume of this

enzyme portion, the monolayer becomes instanta-

neously more condensed. Interestingly, the kinetics

of adsorption of a soluble globular protein as b-

lactoglobulin [32] does not exhibit this kind of

behavior, that is, the surface pressure of a pre-

formed DPPC monolayer only increases smoothly

independent of the initial surface pressure uponthe protein penetration. Finally, the system evolves

to a lower energy state in which the polypeptide

moiety remains at the interface below the polar

groups of the DMPA and the anchor, but in a

more up right position than at lower pi. The

possibility of such a changes in the protein

orientation at the air/liquid interface at high

compression state is likely with the equilibriumsurface pressure�/area curve. This is shown in the

DSAP/DMPA curve in which a transition can be

observed at 20 mN/m to a state that exhibits a

lower isothermal surface compressibility (see Fig.

1A).

Another event that probably can explain the

modification of the profiles in Fig. 4B could be the

sudden DSAP domain formation after the proteininjection into the subphase. Taking into account

that these experiments were carried out upon

injection of the protein in a region approximately

2 mm below the interface, it is likely that DSAP

reaches the interface faster than it can diffuse in

the bulk parallel to the monolayer. Due to the

presence of the anchor the penetration of the

protein at the interface is favorable and it caninstantaneously force the monolayer compression

acting as a ‘‘piston’’ or forming domains. Such

fact, therefore, could also provide the sudden

surface pressure increase observed in the kinetic

curves from Fig. 4B. In this way, the subsequent

surface pressure decrease can be attributed to a

relaxation effect in which the protein molecules

diffuse themselves at the interface among theDMPA molecules. The domain formation upon

protein dispersion could be detected by micro-

scopy techniques and can be object of posterior

studies. Anyway, the relaxation effect associated

to a possible disappearance of protein domains,

could explain the posterior smooth decrease in the

surface pressure.

5. Conclusions

The present study reports two important find-ings. The presence of phospholipid facilitates the

adsorption of DSAP, but not of PLSAP, at the air/

liquid interface. The major contribution to the

dipolar moment of DMPA/DSAP monolayers

results from the phospholipid chain orientation,

in contrast to that of DMPA/PLSAP in which

there is an important contribution of the aminoa-

cid moiety. DSAP exhibits penetration phenomenain DMPA monolayers, forming mixed monolayers

that can be transferred to solid substrates. Thus

DSAP can be immobilized on solid substrates by

adsorption from solution or by the LB technique.

The hydrophobic GPI anchor allows easier ad-

sorption of DSAP to the lipid monolayer in

contrast to PLSAP, which requires some changes

in order to be adsorbed at air/liquid interface. Inspite of the occurrence of interactions between the

enzyme and the polar head group of the phospho-

lipid, the adsorption of PLSAP on solid substrates

could be facilitated through adsorption from

solution mediated by a polar head group, such as

divalent ions. These noticeable differences between

these two solubilized forms should influence

directly the surface density as well as the orienta-tion and enzymatic activity of the immobilized

enzymes in solid substrates.

Acknowledgements

The authors thank CNPq and FAPESP for the

financial support. We also thank Dr Richard J.

Ward and Dr Hector F. Terenzi for careful read-

ing of the manuscript. L. Caseli thanks FAPESP


for the Ph.D. fellowship. F.A. Leone received aresearch scholarship from CNPq.

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