Journal of Oleo Science Copyright ©2016 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : April 15, 2016 doi : 10.5650/jos.ess15247 J. Oleo Sci. Impact of a Reducing Agent on the Dynamic Surface Properties of Lysozyme Solutions Michael M. Tihonov, Viktoria V. Kim and Boris A. Noskov＊ Institute of Chemistry, Saint-Petersburg State University, Saint-Petersburg, 198504, RUSSIA Abstract: Disulfide bond shuffling in the presence of the reducing agents dithiothreitol (DTT) or β- mercaptoethanol (BME) strongly affects the surface properties of lysozyme solutions. The addition of 0.32 mM DTT substantially alters the kinetic dependencies of the dynamic surface elasticity and surface tension relative to those of pure protein solutions. The significant increase in the dynamic surface elasticity likely relates to the cross-linking between lysozyme molecules and the formation of a dense layer of protein globules stabilized by intermolecular disulfide bonds at the liquid/gas interface. This effect differs from the previously described influence of chaotropic denaturants, such as guanidine hydrochloride (GuHCl) and urea, on the surface properties of lysozyme solutions. If both chaotropic and reducing agents are added to protein solutions simultaneously, their effects become superimposed. In the case of mixed lysozyme/GuHCl/ DTT solutions, the dynamic surface elasticity near equilibrium decreases as the GuHCl concentration increases because of the gradual loosening of the cross-linked layer of protein globules but remains much higher than that of lysozyme/GuHCl solutions. Key words: lysozyme, dithiothreitol, guanidine hydrochloride, protein unfolding, dilational surface rheology 1 INTRODUCTION Disordered or partly disordered protein structures are widespread in various biological and industrial systems1－4）. However, these structures have only recently become the subject of intensive study, and the obtained information remains quite limited. Several factors lead to the destruction of protein tertiary Fig. 1 Structural formula of dithiothreitol. structure, such as high pressure and temperature, low pH, and high concentrations of special denaturing substances5, 6）. The use of different denaturing agents can result in differ- shuffle（become disrupted and then undergo random re- ent protein-denaturation mechanisms. combination）5）. Substances able to destroy protein secondary and tertia- The surface properties of protein solutions have been in- ry structures can be divided into two main groups. The vestigated less frequently than their bulk properties first includes chaotropic denaturants, such as guanidine because of the limited number of suitable experimental hydrochloride（GuHCl）and urea. These substances mainly techniques available. Although the interactions between affect hydrogen bonds at the surface of protein globules proteins and denaturants in the surface layer are known to and hydrophobic interactions between different amino acid strongly influence the surface properties11－14）, the details residues but do not affect the molecule’s disulfide bonds5）. of these interactions remain unknown. Most studies investigating the interactions between pro- Recently, the dilational surface rheology was shown to teins and chemical denaturants have focused on systems provide additional information on the protein conforma- containing GuHCl or urea7－12）. The second group comprises tions at the liquid/gas interface15－18）. This approach is reducing agents, such as dithiothreitol（DTT, Fig. 1）and based on the strong difference between the kinetic depen- β-mercaptoethanol（BME）. These substances attack the di- dencies of the dynamic surface elasticity of solutions of sulfide bonds of protein molecules and cause them to globular protein and those of solutions of non-globular or ＊Correspondence to: Boris A. Noskov, Department of Colloid Chemistry, Institute of Chemistry, Saint-Petersburg State University, Saint-Petersburg, 198504, RUSSIA E-mail: [email protected] Accepted December 5, 2015 (received for review October 26, 2015) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1 M. M. Tihonov, V. V. Kim and B. A. Noskov unfolded proteins. In the former case, the kinetic depen- being added to the protein solutions. The volume of the so- dencies are monotonic and resemble the corresponding lution was then increased to the required value. results for aqueous dispersions of charged solid nanoparti- The pH of all solutions was adjusted to 7 by adding com- cles. In the latter case, the kinetic dependencies are similar ponents of the Na2HPO4–NaH2PO4 buffer system（Sigma-Al- to those for solutions of amphiphilic polymers in which the drich Chemie GmbH, Riedstrasse 2 D-89555 Steinhelm 49 dynamic surface elasticity goes through a local maximum 7329 970, Germany）. The ionic strength of all solutions and approaches relatively low values near equilibrium, in was 0.04 M. The solutions were prepared using triply dis- agreement with the theory of the surface viscoelasticity of tilled water. A glass apparatus was used in the last two polymer solutions18）. steps of the distillation. The surface tension of the pure In this work, the surface rheological properties of mixed buffer solution was 72.8 mN/m. solutions of lysozyme, DTT, and GuHCl were studied for All lysozyme solutions were used without storage, and the first time. Although lysozyme is one of the most fre- measurements of the surface properties were started quently studied model proteins, information on the struc- within several minutes after preparing a fresh solution. All ture of its adsorption layers at the liquid/gas interface measurements were performed at 20±1℃. remains rather controversial19－23）. Lysozyme forms small The dynamic dilatational surface elasticity was measured rigid globules in aqueous solutions and belongs to the using the oscillating ring method28, 29）. The surface of the group of“ hard” proteins24）. It has a molecular weight of solution under investigation was periodically expanded and 14,300 Da and consists of 129 amino acid residues. Closed- compressed by the oscillations of a glass ring along its axis. packed lysozyme globules are stabilized by four disulfide The ring was partly immersed into the liquid with its axis bonds, have dimensions of 4.5×3.0×3.0 nm, and consist perpendicular to the liquid surface, and its internal surface of two main domains25）. Lysozyme structure is relatively was grounded to improve wetting. The ring oscillations led stable against high temperature7） and chaotropic chemical to regular oscillations of the liquid surface area and the denaturants, such as urea and GuHCl7－9）, probably because surface tension of the solution because of periodical of the disulfide bonds connecting the remote amino acid changes of the meniscus shape at the internal surface of residues. The presence of these bonds between the 6th and the ring. The surface tension of the investigated liquid was 127th residues and between the 30th and 115th residues measured inside the ring using the Wilhelmy plate method. makes the globule more rigid26）. The addition of a reducing The main advantage of the oscillating ring technique is that agent significantly changes the lysozyme’s globular struc- it creates almost pure dilational deformations of the liquid ture. Chang et al. described the formation of lysozyme surface and therefore contributes negligible shear stresses isomers after the addition of DTT or BME and detected to the experimental results. The relative amplitude and many more unfolded globules in the solutions containing frequency of the solution surface area oscillations were both chaotropic and reducing agents than in those contain- 10％ and 0.1 Hz, respectively. 26） ing only a chaotropic denaturant . The real ε r and imaginary ε i components of the dilational The main aim of this work was to determine the influ- dynamic surface elasticity ε were calculated from the am- ence of lysozyme-denaturant interactions in the surface plitudes of the oscillations of the surface tension δ γ and layer and disulfide bond shuffling on the dilational surface surface area δS and the phase shift φ between the oscilla- rheological properties and obtain additional information on tions of these two quantities by the following relation: protein conformations at the liquid/gas interface. Another dγ Sδγ Sδγ ε＝ ＝ε ＋iε ＝ cos φ＋i sin φ （1） aim of this work was to compare the surface rheological d ln S r i δS δS properties of lysozyme solutions containing a reducing The imaginary part of the complex dynamic surface elastic- agent with those of lysozyme solutions containing a strong ity of the investigated solutions was much smaller than the chaotropic denaturant（GuHCl）studied previously27）. real part. Therefore, only the results for the real part are discussed below. The experimental errors of the oscillating ring method are mainly determined by the errors in the surface tension measurements and are less than ±5％. 2 EXPERIMENTAL PROCEDURES Furthermore, the surface dilational rheology measure- Lysozyme（Sigma-Aldrich Chemie GmbH, Riedstrasse 2 ments were repeated a few times to minimize the random D-89555 Steinhelm 49 7329 970, Germany）was used as re- error. ceived. Lysozyme solutions of required concentrations in phosphate buffer at pH 7 were prepared by diluting a 0.05-mM solution. DTT and GuHCl（Sigma-Aldrich Chemie GmbH, Riedstrasse 2 D-89555 Steinhelm 49 7329 970, 3 RESULTS AND DISCUSSION Germany）were used