a new and simple procedure for the evaluation of the association of surfactants to proteins

8/12/2019 A New and Simple Procedure for the Evaluation of the Association of Surfactants to Proteins

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.J. Biochem. Biophys. Methods 50 2002 261268www.elsevier.com r locate r jbbm

A new and simple procedure for the evaluation of the association of surfactants to proteins

Eduardo Lissi a,) , Elsa Abuin a, Mara E. Lanio b, Carlos Alvarez ba Facultad de Qumica y Biologa, Uni ersidad de Santiago de Chile, Casilla 40-Correo 33, Santiago, Chile

b Laboratorio de Biomembranas, Facultad de Biologa, Uni ersidad de La Habana, Cuba

Received 12 April 2001; received in revised form 10 September 2001; accepted 12 September 2001

Abstract

A new and simple method useful for the evaluation of the association of surfactants to proteinsis proposed. The method is based on an analysis of the effect promoted by surfactant additionupon the fluorescence intensity of the intrinsic tryptophan chromophore and its dependence withprotein concentration. The proposed methodology is applied to quantify the binding of an anionic . Xsodium dodecylsulfate , a zwitterionic N -hexadecyl- N , N -dimethyl-3-ammonio-1-propane-

. . .sulfonate and a neutral Triton X-100, reduced surfactant to bovine serum albumin BSA .q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Surfactants, association to proteins; Bovine serum albumin; Sodium dodecylsulfate; N -hexadecyl-X . N , N -dimethyl-3-ammonio-1-propane sulfonate; Triton X-100 reduced

1. Introduction

The interaction of hydrophobic and amphiphilic molecules with water soluble pro-w xteins and enzymes is a matter of current interest 113 . The association of the solute

can change the secondary and r or tertiary protein conformation, therefore modifying thefunction of the macromolecule. As part of our studies on the relationship betweenconformation and function in hemolytic toxins, we have started a study of the interaction

.of different surfactants with two hemolysins St I and St II obtained from a CaribbeanSea anemone. In order to obtain this information, we have developed a new procedure

)Corresponding author.

. E-mail address: [email protected] E. Lissi .

0165-022X r 02r $ - see front matter q 2002 Elsevier Science B.V. All rights reserved. .PII : S0165-022X 01 00237-8


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( ) E. Lissi et al. r J. Biochem. Biophys. Methods 50 2002 261268 262

for the evaluation of the surfactant association to proteins. To test this procedure, wehave applied it to the association of surfactants to a well known protein, bovine serum

.albumin BSA .The association of a surfactant to a protein can be evaluated by several procedures. In

.most cases, unproved or doubtful assumptions must be done in order to obtain thefraction of the surfactant bound to the protein. Among the most frequently employed

w xmethodologies are those based on surface tension measurements 1,2 , dialysis equilib-w xrium techniques 3 , potentiometric titration through the use of surfactant selective

w x w xelectrodes 4,12 , conductometric titration 5 and spectroscopic measurements based onw x w xthe absorption 5 , and r or emission from either the intrinsic protein chromophores 68

w xor extrinsic fluorescence probes 9,12 . These techniques have several drawbacks.Dialysis equilibrium techniques are time-consuming, while surfactant selective elec-trodes are not simple to prepare for most surfactants and cannot be easily employed at

.very low micromolar surfactant concentrations. Surface tension measurements can becomplex due to the possibility of surface activity of the protein and r or the protein

w xsurfactant complexes 1 . Spectroscopic measurements provide promising approachesdue to the sensitivity of the absorption and r or emission characteristics to the proteinconformation. However, as generally applied, these methodologies require to know thetype of quantitative relationship existing between the absorption of emission propertyconsidered and the amount of bound surfactant. This relationship is usually not known apriori and, for macromolecules, it can be particularly complex. It must be remarked thatspectroscopic methods, as generally used, i.e., at a single protein concentration, can bedirectly applied only when the measured property changes linearly with the solute

w xbinding extent 14 .We have developed a general methodology to measure the partitioning of solutes

w xbetween the dispersion medium and different types of microaggregates 1517 . Themethod is based on the measurement of any property of an intrinsic or extrinsic

.molecular probe or the microaggregate that is modified by the solute. If thesemeasurements are carried out as a function of the solute concentration, at differentamounts of the microaggregates, the distribution of the solute between the microaggre-gates and the external medium can be derived with the only assumption that the effectobserved is only determined by the number of solute molecules associated to each

microaggregate. The method can be employed for any microaggregate, any property, andis independent of the type of dependence that the property presents regarding themicroaggregate concentration. Furthermore, it does not require an a priori assumption of

.the mechanism partitioning or binding by which the solute interacts with the microag-gregate. In previous works, we have applied this methodology to quantify the distribu-

w x w x w xtion of solutes in micelles 15 , reverse micelles 16 , vesicles 15 , and biologicalw xmembranes 17 .

In the present communication, we extend the proposed methodology to the evaluationof the association of surfactants to proteins employing as property the intensity of the

intrinsic fluorescence from protein chromophores. The method is applied to quantify the . Xbinding of an anionic sodium dodecylsulfate , a zwitterionic N -hexadecyl- N , N -di-

. .methyl-3-ammonio-1-propane-sulfonate and a neutral Triton X-100, reduced surfac-tant to bovine serum albumin.


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2. Materials and methods

. XSodium dodecylsulfate, SDS BDH, specially pure , N -hexadecyl- N , N -dimethyl-3- . . .ammonio-1-propane-sulfonate, HPS Sigma , Triton X-100 reduced Aldrich and fatty

.acid-free bovine serum albumin, BSA Sigma were used as received. Water employed .was conductivity grade purified by a Millipore Milli-Q water purification system.Fluorescence spectra and fluorescence intensities were recorded on a Spex Fluorolog

1681 fluorescence spectrometer. The tryptophan chromophore was excited at 295 nmand fluorescence spectra were recorded from 310 to 450 nm. A 10-mm path lengthquartz cell was used. Excitation and emission slit widths were 1.25 nm.

All experiments were carried out at 22 8C. The pH of the solutions, registered beforeand after addition of the highest surfactant concentration employed, was in the range5.56.0 for all the surfactants considered.

3. Results and discussion

.Addition of SDS, HPS or Triton X-100 reduced to BSA solutions produce adecrease in the intensity of the tryptophan fluorescence concomitant with a blue shift of the emission maximum. The magnitude of the effect, quantified in terms of the ratio

0 0 I r I I and I being the fluorescence intensities, registered at a given wavelength, in.the absence and presence of the surfactant considered, respectively , reaches a plateau at

relatively low surfactant concentrations. Results obtained working at a single BSAconcentration equal to 8 mM are shown in Fig. 1. The data given in this figure show that

0 0 .both the I r I values reached the plateau, I r I , and the amount of surfactantplateau0 0 .required to reach an I r I equal to 0.5 I r I are dependent on the surfactantplateau

considered. However, these data, obtained at a single protein concentration, do not allowfor an evaluation of the binding efficiency. To quantify the amount of association of each surfactant to the protein, it is necessary to look for the dependence of the I 0r I vs.w x w xsurfactant profiles with the protein concentration 15,16 .

The amount of surfactant required to each given I 0r I value is displaced towardshigher concentrations when the concentration of the protein is increased. Representativeresults obtained using HPS are shown in Fig. 2. Similar profiles were obtained usingeither SDS or Triton X-100.

0 w xThe effect of BSA concentration on the I r I vs. surfactant profiles is attributed tothe surfactant distribution between the protein and the external medium. The distributionof the solute can be quantified in terms of a pseudo-binding constant, K , according tob

.Eq. 1 ,

w x w x w xK s Surf r Surf BSA 1 4 .b freeb

w xwhere Surf is the analytical concentration of surfactant associated with the protein,bw x w xSurf is the concentration of surfactant remaining in the external medium and BSAfree


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0 Fig. 1. Effect of surfactant addition on the tryptophan fluorescence intensity ratio, I r I see text for.definition , in a BSA solution at 8 mM concentration. Excitation wavelenght s 295 nm. Fluorescence

. . . .wavelenght s 342 nm. Added surfactant: SDS v ; HPS B and Triton X-100 reduced % .

w x w xis the protein concentration. Since Surf r BSA is the average number of surfactantbmolecules, n, bound per mol of BSA, K can be further expressed asb

w xK s nr Surf 2 .freebThe method proposed to evaluate K is based on the assumption that the I 0r I valueb

is only determined by the average number of surfactant molecules bound to each protein . w x ..n . Since equal n implies equal Surf from Eq. 2 , a simple mass balance leads tofree

w x w x w xSurf s Surf q n BSA 3 .analyt freew x w x w xwhere Surf is the total surfactant concentration. For a set of Surf and BSAanalyt analyt

0 .values corresponding to the same I r I , a plot of the left hand side of Eq. 3 againstw xBSA allows for the evaluation of K from the slope r intercept ratio. Results such thatb

.shown in Fig. 2 and similar results obtained using SDS or Triton X-100 reduced weretreated according with to procedure. Plots obtained for a set of data taken at I 0r I s 1.3

. 5are shown in Fig. 3. From the lines of this plot, values of K s 0.47 " 0.11 = 10by 1 . 5 y 1 . 5 y 1M , K s 0.65 " 0.40 = 10 M and K s 0.085 " 0.004 = 10 M were ob-b b

.tained for the binding of HPS, SDS and Triton X-100 reduced to BSA, respectively.The values of K determined by the proposed procedure show noticeable differencesbregarding the errors involved in their determinations. While for HPS and Triton X-100,

the values are relatively accurate, the result obtained for SDS is bound to a very largeerror. This is due to the fact that the amount of surfactant remaining in the externalmedium is small and hence bound to a large relative error. This is emphasized by the

.data given in Table 1 that summarizes the values of n derived from the slopes and


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0 Fig. 2. Effect of BSA concentration on the tryptophan fluorescence intensity ratio, I r I see text for.definition , promoted by addition of HPS. Excitation wavelength s 295 nm. Fluorescence wavelength s 342

. . . .nm. BSA concentrations: 1 mM v ; 4 mM ' ; 8 mM % and 15 mM B .

w x .Surf derived from the intercepts obtained for the three surfactants considered. Thefreew xdata show that, while absolute errors are relatively small, the relative error in Surf free

for SDS is extremely large. This large error is reflected in the large uncertaintyassociated with the K value for SDS. This implies that the proposed method is able tobprovide accurate values of the amounts of free and bound surfactant but it gives largeerrors for the K evaluation for cases in which there remains only a small fraction of thebsurfactant in the external medium.

The low value of n required to produce a noticeable change in the intrinsic

fluorescence intensity of the protein is remarkable. In fact, the data given in Table 1show that for SDS, 1.84 surfactant molecules per protein leads to an I 0r I value of 1.3.This is ca. 50% of the total change observed at saturation. This extreme sensitivityimplies that the binding is evaluated at very low surfactant concentrations. This makesdifficult a comparison of the present data with data obtained, by other procedures, at

w xconsiderably higher surfactant concentrations 5 . On the other hand, it allows anevaluation of the binding extent under conditions such that the free surfactant concentra-

tion is considerably lower than the corresponding critical micellar concentration 8 mM. w xfor SDS, 90 mM for HPS and 360 mM for reduced Triton X-100 18,19 .

The binding of solutes to proteins is complex and usually cannot be represented byw xisotherm and r or a single binding constant 5,2022 due both to interaction between

sites and r or changes in the protein conformation as a consequence of the solute binding.One of the advantages of the proposed procedure is that its application is general and


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.Fig. 3. Experimental data of Fig. 2 and similar results obtained using SDS or Triton X-100 reduced , plotted . 0 . . . .according to Eq. 3 . Set of data taken at I r I s 1.3; v SDS; B HPS; % Triton X-100 reduced .

.does not requires to know or postulate the analytical function linking the amount of solute adsorbed to the free solute concentration. Also, it is independent of the type of

relationship existing between the measured property the fluorescence intensity in the.present work and the number of solute molecules bound per protein.

The data treatment described above, when applied to sets of data taken at different0 I r I values, allows an estimation of the dependence of K with n or the concentrationb

. w xof free surfactant 15,16 . This allows to determine if the binding process presents a .significant cooperativity increase in K with n or an anti-cooperative or saturationb

.behaviour decrease in K with n . This type of analysis has been performed for thebHPS data. The results given in Table 1 would indicate that, at the low n values

Table 1Values of the average number of surfactant molecules bound per mol of BSA, n, the concentration of freesurfactant, and the apparent binding constant at the indicated I 0 r I values

0 y 1w x . .Surfactant I r I n Surf mM K mMfree bSDS 1.30 1.84 " 0.15 2.84 " 1.6 0.65 " 0.40

Triton X-100 1.30 4.10"

0.11 48"

1 0.085"

0.004HPS 1.15 2.61 " 0.07 4.5 " 0.7 0.58 " 0.101.30 5.20 " 0.25 11 " 2 0.47 " 0.111.45 8.8 " 0.1 25 " 1 0.35 " 0.03


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considered in the present work, there is not indication of cooperativity and that there is aclear tendency to a decrease in the apparent binding constant when the free surfactantconcentration increases. This decrease could be due to an electrostatic effect, operativeunder conditions where there is no significative cooperativity associated to the formationof micelle-like aggregates on the protein.

Acknowledgements

.Financial support of this work by Dicty USACH and a collaborative Project . .CONICYT Chile r CITMA Cuba is acknowledged.

References

w x1 Tribout M, Paredes S, Gonzalez-Manas JM, Goni FM. Binding of Triton X-100 to bovine serum albumin as studied by surface tension measurements. Biochem Biophys Methods 1991;22:12933.

w x2 Folmer B, Holmberg K, Svensson M. Interaction of Rhizomucor miehei lipase with an amphotericsurfactant at different pH values. Langmuir 1997;13:58649.

w x3 Hiramatsu K, Ueda Ch, Iwata K, Arikawa K, Aoki K. The interaction of bovine plasma albumin withcationic detergent: studies by binding isotherm, optical rotation and difference spectrum. Bull Chem SocJpn 1977;50:36872.

w x4 Hiramatsu K, Yang JT. Cooperative binding of hexadecyltrimethylammonium chloride and sodiumdodecylsulfate to cytochrome c and the resultant change in protein conformation. Biochim Biophys Acta1983;743:10614.

w x5 Takeda K, Miura M, Takagi T. Stepwise formation of complexes between sodium dodecylsulfate andbovine serum albumin detected by measurements of electric conductivity, binding isotherm and circulardichroism. J Colloid Interface Sci 1981;82:3844.

w x6 Johansson JS. Binding of the volatile anesthetic chloroform to albumin demonstrated using tryptophanfluorescence quenching. J Biol Chem 1997;272:179615.

w x7 Edwards K, Chan RYS, Sawyer WH. Interactions between fatty acids and lipoprotein lipase: specificbinding and complex formation. Biochemistry 1994;33:1330411.

w x8 He B, Zhang Y, Wang H-R, Zhon H-M. Inactivation and unfolding of aminoacylase during denaturationin sodium dodecylsulfate solutions. J Protein Chem 1995;14:34957.

w x9 Avdulov NA, Chochina SV, Davagan VA, Shroederer F, Mayo KH, Wood WG. Direct binding of ethanolto bovine serum albumin: a fluorescent and 13 CNMR multiplet relaxation study. Biochemistry 1996;35:3407.

w x10 Abuin E, Lissi E, Godoy X. Independent and simultaneous effects of urea and sodium dodecylsulfate onthe enzymatic activity of catalase. Bol Soc Chil Quim 1999;44:1239.

w x11 Henriquez M, Abuin E, Lissi E. Effects of urea and sodium dodecylsulfate on the formation of microdomains in aqueous solutions of gelatin. J Colloid Interface Sci 1996;179:5326.

w x12 Henriquez M, Abuin E, Lissi E. Interactions of ionic surfactants with gelatin in fluid solutions and the gelstate studied by fluorescence techniques, potentiometry and measurements of viscosity and gel strength.Colloid Polym Sci 1993;271:9606.

w x13 Moriyama Y, Takeda K. Re-formation of the helical structure of human serum albumin by the addition of small amounts of sodium dodecylsulfate after disruption of the structure by urea: a comparison withbovine serum albumin. Langmuir 1999;15:20038.

w x14 Lissi E, Gunther G, Zanocco AL. Evaluation of association constants from changes in the shape of fluorescence spectra: a corrected method. Photochem Photobiol 1996;63:70911.

w x15 Abuin E, Lissi E. In: Christian SD, Scamehorn JF, editors. Solubilization in surfactant aggregates. NewYork: Marcel Dekker; 1995. p. 297332.


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a new and simple procedure for the evaluation of the association of surfactants to proteins

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