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Colloids and Surfaces A: Physicochem. Eng. Aspects 298 (2007) 88–93

Advantages of interfacial tensiometry for studying the interactionsof biologically active compounds

Alla N. Generalova a,b,∗, Svetlana B. Marchenko a,b, Irina V. Gorokhova a,b, Reinhard Miller b,Irina V. Gurevich b,c, Marina S. Tsarkova c, Vladimir I. Maksimov c, Sergei Yu. Zaitsev a,c

a Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russiab Max-Planck-Institut fur Kolloid und Grenzflachenforschung, Max-Planck-Campus, Am Muhlenberg 1, D-14476 Golm, Germany

c Moscow State Academy of Veterinary Medicine and Biotechnology, Acad. Skryabina, Str. 23, Moscow 109472, Russia

Available online 15 December 2006

bstract

Dynamic surface tensions of lipase in aqueous solutions at pH 7, in Na-phosphate solution at pH 9 and in basic phosphate buffer at pH 11 wereeasured at the water/air interface in a broad concentration range (10−8 to 10−5 M). Different lipase preparations were immobilized under various

onditions in polyelectrolyte complexes (PEC) based on Na-poly(styrene sulfonates) (PSS) as polyanion and poly(diallyldimethylammoniumhloride) (PDADMAC) as polycation. Strong lowering of the surface tension was observed for both two component and three component complexes.he dynamic surface tension of these systems allowed conclusions about the stability of the highly surface-active polyelectrolyte complexes whichere formed under well defined conditions—pH value during complex formation, ionic strength, polyelectrolyte charge and polyelectrolyte ratio. 2006 Elsevier B.V. All rights reserved.

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eywords: Lipase; Polyelectrolyte complex; Immobilized enzyme; Dynamic su

. Introduction

Recently surface tension measurements of biologically activeiquids are widely used in biochemistry, food testing, medicalnd environmental research and other fields [1]. This promisingiagnostic method can provide important information both fromtechnological point of view and to understand the respective

dsorption mechanisms [2].Among the various well-known techniques, the drop and

ubble profile analysis tensiometry is the most frequentlysed. The main advantages of this absolute method is theery small amount of sample that is required, making theethod applicable to any liquid interface. In addition, the high

eproducibility and computer controlled data acquisition andandling make the experiment relatively straightforward to per-

orm [3].

The aim of the present work is the general study of lipasemmobilization in polyelectrolyte complexes (PEC), in particu-

∗ Corresponding author at: Institute of Bioorganic Chemistry, Russiancademy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russia.

E-mail addresses: alng@zmail.ru (A.N. Generalova),iller@mpikg-golm.mpg.de (R. Miller), szaitsev@mailru.com (S.Yu. Zaitsev).

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927-7757/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2006.12.014

tension; Drop profile analysis tensiometry

ar, investigations of the complex formation via dynamic surfaceension measurements at the water/air interface.

Lipase was chosen for this study as one of the best studiedydrolases, which can catalyze four different reactions, such asydrolysis of lipids and esters, esterification, transesterificationnd acylic group transition. Moreover, the well-known abilityf lipase activation at the interfaces and by immobilization onydrophobic carriers, is another important target of the presentork [4].One of the most important field of modern biochemistry

nd biotechnology is the design of compositions and com-lexes, combining synthetic and natural polymers, in particularighly effective “biocatalytic” or “biosensoring” polymer mate-ials (obtained by enzyme immobilization and modification) [5].harged polyelectrolytes are the most suitable polymeric mate-

ials for physical immobilization of enzymes in a polymer matrixue to electrostatic interactions [6]. A variety of polyelectrolyteomplexes can be obtained by changing the chemical structuref polymers, such as molecular weight, flexibility, functionalroup structure, charge density, hydrophilicity–hydrophobicity

alance, stereoregularity and compatibility, as well as reac-ion conditions (pH, ionic strength, concentration, mixing rationd temperature) [7]. Enzyme adsorption on charged polyelec-rolytes is one of the main methods of physical immobilization of

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One can see that the addition of lipase decreased the surfacetension of PSS aqueous solution down to 50 mN/m. In this case itis likely that a surface-active complex was formed which lowers

A.N. Generalova et al. / Colloids and Surfaces

rotein in polymer matrices. The principal advantages of suchmmobilization are the increasing stability of the enzymes inime and to various denaturation conditions, obtaining suitable

aterials for technical applications, effective separation fromhe reaction media and multiple usage [8]. It was shown, that thectivity of immobilized enzyme strongly depends on the prepa-ation conditions, i.e. pH value during complex formation, theolyelectrolyte/enzyme concentration ratio and the molar massf the polyelectrolyte [7,9].

Different lipase preparations were immobilized at vari-us conditions in polyelectrolyte complexes—Na-poly(styreneulfonates) (NaPSS) (as polyanion) and poly(diallyldimethyl-mmonium chloride) (PDADMAC) (as polycation). In ournvestigations we chose reagent concentrations (lipase, poly-lectrolyte and ratio of polymers in complexes) previouslyharacterized by light scattering analysis and lipase activityeasurements [9].

. Experimental

For preparation of the polyelectrolyte–enzyme complexes,a-poly(styrene sulfonates) with Mw = 70,000 as polyanion andoly(diallyldimethylammonium chloride) with Mw = 300,000 asolycation were purchased from Fluka. All salts were purchasedrom Sigma. Lipase, crude powder of the lipase from hog pan-rease, Mw = 50,000, pI = 5.18 and purity of 20%, was purchasedrom Fluka. The lipase powder was dissolved in water, in Na-hosphate salt (0.01 M Na2HPO4, pH 9), in basic phosphateuffer (50 ml 0.05 M Na2HPO4 with 4.1 ml 0.1 M NaOH in00 ml H2O, pH 11).

The previously obtained data (static light scattering measure-ents) [9] were taken into account to prepare both the lipase

olutions of concentrations in the range 10−5 to 10−8 M, andipase:PSS with ratios 1:50 and 1:100, polyelectrolyte com-lexes (PSS:PDADMAC) with ratios 1:0.3, 1:0.6 and 1:1.

The complexes with lipase were prepared in two steps. Therst step was gentle mixing of the lipase solution with the firstolyelectrolyte (having the same kind of charge as lipase at thearticular pH). The second step started with the preparation ofhe solution of the second polyelectrolyte, and then this solutionas mixed with the solution containing lipase—first polyelec-

rolyte in amounts of 1 ml each until the desired mixing ratioas reached.The studied systems at the three pH values 7, 9 and 11 were:

1) Lipase as typical enzyme;2) Na-poly(styrene sulfonates) as polyanion;3) lipase with polyanion (PSS);4) poly(diallyldimethylammonium chloride) as polycation;5) lipase with polycation (PDADMAC);6) mixture of polyanion and polycation (PSS:PDADMAC);7) complexes—lipase with mixture of polyanion and polyca-

tion (PSS:PDADMAC).

The surface tensions of the mentioned complexes were mea-ured by using the pendent drop technique The main principlef this technique (drop profile analysis) is to determine the sur-

F9

hysicochem. Eng. Aspects 298 (2007) 88–93 89

ace tension of a liquid from the shape of a pendent drop oruoyant bubble. The instrument PAT-1 (SINTERFACE Tech-ologies, Berlin, Germany) is based on this principle and allowsontinuous measurement of the surface or interfacial tension asfunction of time while the surface area can be kept constant.he analysis of results and all functionalities are computer con-

rolled. After forming the drop of the final mixture by the dosingystem 5 min were required to ensure a stability of drop. We usedhe water–air interface in all measurements. The experimentalemperature was controlled at 22 ◦C [10].

. Results and discussion

Dynamic surface tensions of lipase solutions in water (pH 7),a-phosphate (pH 9) and basic phosphate buffer (pH 11) at dif-

erent concentrations (10−8 to 10−5 M) were measured (Fig. 1).t the studied concentrations, the surface tensions of lipase solu-

ions was significantly decreased (down to 45 mN/m) with timenly at maximal concentration in pH 9 and 11, and some changesan be marked at concentration 10−6 M in these solutions. Forurther investigations the first concentration with no changing ofhe surface tension in all solutions which can detect changing ofhe complex surface tension more precisely was chosen. It was0−7 M.

Previously [9] it was found that effective complexes of lipaseith PSS were formed at ratios lipase:PSS of 1:50 and 1:100,

o dynamic surface tensions of PSS were measured at concen-rations 5 × 10−6 (I) and 10−5 M (II), which correspond to thebove referred ratios at all pH (Fig. 2). It is worth noticing that allSS solutions at pH 9 and 11 had identical regularity and the sur-ace tension was decreased down to 50–55 mN/m, while in waterhe surface tension was practically constant. Probably, the bufferalt addition screened the electrostatic interaction [7] in PSSolution which resulted in a change of the hydrophilic–lipophilic

ig. 1. Surface tension of lipase solutions in water (pH 7), Na-phosphate (pH) and buffer (pH 11) depending on concentration.

90 A.N. Generalova et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 298 (2007) 88–93

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ig. 2. Dynamic surface tension of PSS solutions at different pH at concentra-ions I and II.

he surface tension. The presence of lipase in PSS solutionst increasing pH (in Na-phosphate and buffer) decreased theurface tension the more the higher the pH (Fig. 3), but not asuch as in the case of the aqueous solutions. Probably, in these

ases we have a weaker complex formation.Dynamic surface tensions of aqueous drops containing

DADMAC with concentration in respect to PDADMAC:PSSatios of 0.3, 0.6, 1, where PSS were I or II, were measuredt different pH. As one can see the surface tension of PDAD-AC aqueous solution was decreased down to 55–57 mN/m

nly at the concentration in respect to the equimolar ratio (1:1)or both PSS concentration (Figs. 4 and 5). At pH values of 9nd 11 the decrease started from a PDADMAC concentrationorresponding to a ratio of 0.6. For the second PSS concentra-ion the dynamic surface tension of PDADMAC at pH 11 waslowly decreased and only up to a surface tension of 63 mN/mFig. 5). Possibly, the quaternary ammonium compound PDAD-

AC was screened by addition of buffer salt [7] with increasingH which results in a decrease of the PDADMAC surfacectivity.

ig. 3. Dynamic surface tension of different PSS solutions in presence of lipase.

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ig. 4. Surface tension of PDADMAC solutions with concentration in respecto various mixing ratios PDADMAC/PSS at PSS concentration I.

After lipase addition pronounced changes in surfaceension of aqueous PDADMAC solutions were observednly at the highest concentrations (in respect to the ratioSS:PDADMAC = 1:1 for both PSS concentrations) (cf.igs. 6 and 7). In these cases the dynamic surface tensions were

ncreased up to 72 mN/m which is possibly caused by the for-ation of complexes with an almost complete compensation

f positive (PDADMAC) and negative (lipase) charges. WithH increase of the polymer–lipase solutions (in Na-phosphatend buffer) the surface tension decreases down to 45 mN/m,specially at PDADMAC concentrations for the ratio of 0.3.hese data indicate that both lipase and polymer adsorbed at theater/air interface and formed highly surface-active complexes.robably, the high pH level promotes such complex formationue to the increase of negative charge of lipase molecules andnchanged charge of the PDADMAC molecules. It is based onhe well-known fact that stable complexes form in excess of one

f the charges [7].

Dynamic surface tension of polyelectrolyte mixtures PDAD-AC: PSS with ratios of 0.3, 0.6 and 1 in respect to PSS

ig. 5. Surface tension of PDADMAC solutions with concentration in respecto various mixing ratios PDADMAC/PSS at PSS concentration II.

A.N. Generalova et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 298 (2007) 88–93 91

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Fig. 8. Dynamic surface tension of polymer mixed solutions PDAD-MAC/PSS = 0.3 as a function of PSS concentrations I and II at differentpH.

ig. 6. Surface tension of PDADMAC solutions with concentration in respecto various mixing ratios PDADMAC/PSS at PSS concentration I in presence ofipase.

oncentration at all pH were measured (Figs. 8, 9 and 10).s one can see the complexes at a ratio of 0.3 had the mean

urface tension of both polymers. Only the complex in bufferolution for a PSS concentration of 5 × 10−6 M (I) (correspond-ng ratio lipase:PSS was 1:50) showed an increased loweringf surface tension down to 44 mN/m (Fig. 8). Polyelectrolyteomplexes with a ratio of 0.6 displayed the mean surface ten-ion value of both polymers (Fig. 9). The same situation wasbserved for a ratio of 1 (Fig. 10) but additional surface tensionecrease was observed in three cases (lipase:PSS = 1:50 at pH1; lipase:PSS = 1:100 at pH 9 and pH 11).

Three component complexes (lipase with a polyelectrolyteixture) with ratio PDADMAC:PSS = 0.3 decreased the surface

ension of the solutions at pH 9 and 11, but not in water, for allases down to 50 mN/m (Fig. 11). We could observe the same

ituation for complexes with a ratio of 0.6 though the surface ten-ions were less decreased except at pH 11 (lipase:PSS = 1:100)Fig. 12). The dynamic surface tension of the complex solutionsith a ratio of 1 in buffer decreased for both PSS concentrations

ig. 7. Surface tension of PDADMAC solutions with concentration in respecto various mixing ratios PDADMAC/PSS at PSS concentration II in presencef lipase.

Fig. 9. Dynamic surface tension of the polymer mixture solutions PDAD-MAC/PSS = 0.6 as a function of PSS concentrations I and II at differentpH.

Fig. 10. Dynamic surface tension of the polymer mixture solutions PDAD-MAC/PSS = 1 as a function of PSS concentration I and II at differentpH.

92 A.N. Generalova et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 298 (2007) 88–93

Table 1Dynamic surface tension and activity data of the PEC

PEC pH Mixing ratio,CPDADMAC/(Clip + CPSS)

Clip:CPSS Dynamic surface tension of PEC(by extrapolation to infinity)

PEC activitya (% of startinglipase activity 40 U/ml)

1 9 0.3 1:50 50 722 9 0.3 1:100 53 723 9 0.6 1:50 63 674 9 0.6 1:100 57 925 9 1 1:50 57 866 9 1 1:100 50 1257 11 0.3 1:50 52 –8 11 0.3 1:100 48 –9 11 0.6 1:50 57 99

10 11 0.6 1:100 45 10442 131

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a PEC activity was measured by monitoring the hydrolysis of triacetin with a

Fig. 13). These effects can be explained in terms of changes inharge as described before.

The data of the surface-active multi-component complex for-ation (Table 1, rows 10–12) with immobilized lipase are in

ig. 11. Dynamic surface tension of the polymer mixture solutions PDAD-AC/PSS = 0.3 as a function of PSS concentrations I and II at different pH in

resence of lipase.

ig. 12. Dynamic surface tension of the polymer mixture solutions PDAD-AC/PSS = 0.6 as a function of PSS concentration I and II at different pH in

resence of lipase.

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ig. 13. Dynamic surface tension of the polymer mixture solutions PDAD-AC/PSS = 1 as a function of PSS concentration I and II in presence of lipase.

ood accordance with data of lipase enzyme activity measuredy monitoring the hydrolysis of triacetin.

. Conclusion

Stable highly surface-active polyelectrolyte complexes arebtained as shown by drop shape tensiometry under defi-ite conditions—under defined pH conditions during complexormation, ionic strength, polyelectrolyte charge and polyelec-rolyte ratio. Strong lowering of the surface tension was found foroth the two component (lipase:PSS mixed systems with pH 7;ipase: PDADMAC with pH 11; PSS: PDADMAC = 1:0.3 withH 9; PSS: PDADMAC = 1:1 with pH 9 and 11) and three com-onent complexes (lipase:PSS: PDADMAC = 1:0.6 and 1:1 withH 11). The pendent drop profile analysis technique proves toe a suitable tool for the investigation of enzyme immobilizationn polyelectrolyte complexes.

cknowledgement

The work was financially supported by a project of the Ger-an Science Foundation (DFG 436 RUS 113/697/5-1).

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