Coacervates of Lactotransferrin and β- or κ-Casein: Structure

Article pubs.acs.org/Langmuir

Coacervates of Lactotransferrin and β- or κ‑Casein: Structure Determined Using SAXS C. G. (Kees) de Kruif,*,†,∥ JanSkov Pedersen,‡ Thom Huppertz,§ and Skelte G. Anema∥ †

Van’t Hoff laboratory for Physical and Colloid Chemistry, Padualaan 8, Utrecht University, The Netherlands Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Langelandsgade 140, DK-8000 Århus, Denmark § NIZO food research, PO Box 20, 6710 BA, Ede, The Netherlands ∥ Fonterra Research Centre, Private Bag 11029, Dairy Farm Road, Palmerston North, New Zealand ‡

ABSTRACT: Lactotransferrin (LF) is a large globular protein in milk with immuneregulatory and bactericidal properties. At pH 6.5, LF (M = 78 kDa) carries a net (calculated) charge of +21. β-Casein (BCN) and κ-casein (KCN) are part of the casein micelle complex in milk. Both BCN and KCN are amphiphillic proteins with a molar mass of 24 and 19 kDa and carry net charges of −14 and −4, respectively. Both BCN and KCN form soap-like micelles, with 40 and 65 monomers, respectively. The net negative charges are located in the corona of the micelles. On mixing LF with the caseins, coacervates are formed. We analyzed the structure of these coarcervates using SAXS. It was found that LF binds to the corona of the micellar structures, at the charge neutrality point. BCN/LF and KCN/LF ratios at the charge neutrality point were found to be ∼1.2 and ∼5, respectively. We think that the findings are relevant for the protection mechanism of globular proteins in bodily fluids where unstructured proteins are abundant (saliva). The complexes will prevent docking of enzymes on specific charged groups on the globular protein.

I. INTRODUCTION The interaction and self-assembly of oppositely charged proteins is a topic of recent research. Historically, it starts with Tiebackx1 who first reported on the coacervation of gum Arabic (GA) and gelatin. However, it was Bungenberg de Jong and Kruyt2 who made the first systematic investigation into the phenomenon, using a positively charged protein (gelatin) and the negatively charged polysaccharide GA. The interaction of oppositely charged (bio)polymers has an important application in encapsulation. Again, Bungenberg de Jong and Kruyt2 were first to show encapsulation of carbon particles and dyes in the GA/gelatin system. The National Cash Register company developed carbonless copy paper in the early 1950′s based on this system. Nowadays, for encapsulation, the so-called layer by layer technology is often used.3 Recent reviews on the interaction of oppositely charged (bio)polymers are presented by Izmudov4 and in a special issue of Sof t Matter entitled “Polyelectrolytes in biology and soft matter”.5 In Figure 1, we present a schematic illustration of the main types of coacervates. Systems containing a polyelectrolyte/ polysaccharide and an oppositely charged protein or colloid have been extensively researched and were reviewed by De Kruif et al.,6 Schmitt and Turgeon,7 and, more recently, Kayitzmayer.8 In general, complexes and coacervate phases are formed when the two colloids are on the opposite side of the isoelectric point (IEP) and at low(er) ionic strength. Proteins are patchy colloids and they may even form coacervates at the “wrong” side of the © 2013 American Chemical Society

Figure 1. Schematic illustration of the morphology of various coacervates. Red color: positively charged; blue color: negatively charged (or vice versa). A: chromatin, the DNA-histone complex (from Arcesi et al.15,17). B: gum Arabic + β-lactoglobulin (from Weinbreck et al.10). C: gum Arabic + gelatin (from Bungenberg de Jong and Kruyt2). D: BCN + lactotransferrin (from Pan et al.,14 Anema and De Kruif,12 and this paper). E: lysozyme in a polyelectrolyte brush (from Witteman and Ballauf18). F: complex coacervate core micelles (from Voets et al.19) or surfactant micelles + polyeletrolyte (from Chiappisi et al.20). G: ionic crystals made of PMMA spheres (from Leunissen et al.21). H: (fractal) aggregates of lysozyme and β-lactoglobulin (from Howell et al.22) or silica particles (from Kim and Berg,23 Rasa et al.24). Note: drawings are not to scale.

Received: June 17, 2013 Revised: July 16, 2013 Published: July 16, 2013 10483

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IEP.9 Small proteins (colloids) may decorate the polysaccharide as depicted by Weinbreck et al.10 Croguennec and co-workers investigated the formation of hierarchical self-assembly of α-lactalbumin with lysozyme.11 Supramolecular structures are formed from tetrahedral protein building blocks containing two lysozyme and two α-lactalbumin molecules interacting through their oppositely charged patches. Interestingly any further supramolecular assembly seems to be driven by hydrophobic interactions. Anema and De Kruif12,13 studied the interaction between folded proteins (lactotransferrin and lysozyme) with unstructured proteins (e.g., αS-casein). These proteins form coacervates and lactotransferrin forms stable (overcharged) associates on either side of the charge neutrality point. αS-Casein can be considered as poly(ampho)eletrolyte and then there is a great similarity with the coacervation of polysaccharides and oppositely charged colloids or proteins. Pan et al.14 interacted lysozyme with β-casein (BCN) and subsequently denatured the lysozyme. If the proteins (colloids) are large enough, the polyelectrolyte may wind around the protein. For instance, in the chromatin complex, DNA winds around the histones,15 therewith effectively folding the DNA into the cell. The folding comes with an energetic penalty, i.e., folding of the stiff polyelectrolyte chain. Park et al.,16 Arcesi et al.,17 and Anema and De Kruif13 showed that one can add small amounts of lysozyme to BCN (at low salt content) without flocculation; however, adding BCN to lysozyme flocculated the system and it was suggested that this was due to the folding energy penalty of BCN around the very small lysozyme. If the polyelectrolyte forms a brush, e.g., by grafting to the surface of a colloid or because it has a hydrophobic tail, then the proteins are attached to the charged polymer corona, as shown by Witteman and Ballauff,18 and in this work. Voets et al.19 reviewed the work on complex coacervate core micelles in which diblock polymers have a neutral tail, but an oppositely charged head. These systems form coacervates but cannot grow because of the neutral tail. Biesheuvel et al.25 studied the interaction between two folded proteins, i.e., lysozyme and succinylated lysozyme which can be compared with the interaction of oppositely charged colloid particles, e.g., by Kim and Berg23 and Rasa et al.24 The work on oppositely charged colloids shows rich phase behavior, from fractal aggregates to ordered crystalline structures. Both Rasa et al.24 and Leunissen et al.21 used low dielectric solvents, in which the product of the Debye−Huckel screening parameter κ, and particle radius a, is large; i.e., κa ≫ 1. By manipulating the conditions, Leunissen et al.21 were able to mimic the crystalline structures of simple salts. In addition to the long-range attraction the PPMA particles have a short-range polymeric repulsion. In this paper we studied the adsorption of lactotransferrin to the polyelectrolyte brush of BCN micelles. Lactotransferrin (LF) is a large (78 kDa) protein with pI ≈ 8.32 and has immune regulatory and bacteriostatic properties.26,27 BCN is an unstructured protein of 209 amino acids from milk. It is amphiphilic with a molecular mass of 24 kDa and a pI ≈ 5.6. The net number of charges on both proteins can be calculated using the primary sequence of the Swiss Protein Database.28 Early investigations on the temperature driven hydrophobic self-association of BCN were made by Payens29 during the 1960s using light scattering, analytical ultracentrifugation, viscosity measurements, and high pressure measurements. Kegeles30 proposed a so-called shell model for the self-association of BCN. Later, the work on BCN was extended often under varying and different conditions.31,32 Interestingly, Leclerc and Calmettes33

measured second virial coefficients of the micelles and found slightly negative values. This is consistent with the idea that there must be an overall attractive (cooperative) interaction in order to form micelles. Mikheeva et al.34 made an extensive investigation of the thermodynamics of the micellization of BCN confirming the shell model proposed by Kegeles.30,35 The micellization enthalpy is however endothermic, indicating that micellization is strongly entropy driven.36 Thus, BCN is monomeric at temperatures below 5 °C and forms polymeric micelles containing about 40 monomers at temperatures above 30 °C. Details will depend on the buffers used and the addition of “chaotropes”. For example, guanidine hydrochloride or urea will dissociate the BCN micelles. However, cross-linking the micelles using transglutaminase prevents the dissociation and leaves the micelle greatly intact.37,38 From small angle scattering experiments, the size of the micelles in a 25 mM phosphate buffer is about 15 nm (hydrodynamic radius and interaction radius) and the radius of gyration is 6 nm.32 Hence, it was concluded that the micelles had a relatively dense core of about 7 nm radius and a fluffy outside, being the hydrophilic N-terminus of BCN. In a recent paper, the association of BCN (and KCN) was studied using SANS and SLS.39 The (truncated) scattering spectra were fitted to the sum of an oblate ellipsoid and a Debye function, and are generally consistent with previous studies. Interestingly, Ossowski et al.39 studied the fibril formation on standing and applying shear. On increasing the concentration, the BCN dispersions become very viscous, but viscosity decreases exponentially with temperature and increases with ionic strength.40 BCN micelles have been claimed to have chaperone-like properties.41,42 It remains to be seen whether this is a real chaperone property, i.e., preventing proteins from aggregation during unfolding/folding, or just the generic surfactant property of BCN in stabilizing hydrophobic particles like emulsion droplets. Actually, experiments on heating lysozyme in the presence of BCN showed that refolding was inhibited in the presence of BCN.43 We have used caseins to stabilize gold sols and whey protein particles (unpublished results), and BCN is known to absorb small molecules such as vitamins and drugs.44 κ-Casein (KCN) is famous for its stabilization of the casein micelle. Often it is claimed to be of electrostatic origin but that is improbable, because KCN caries only few net charges and, in addition, the Debye−Huckel screening parameter in milk is only 1 nm. A quantitative calculation of the steric, electrostatic, and van der Waals potential was given by Tunier and De Kruif45 showing that electrostatic contributions are small. Numerous publications discuss the interaction of KCN with the (heated) whey proteins or with added polysaccharides. There are few papers on the micellization of KCN. KCN forms micellar structures, and the size and number of monomers (∼30) is largely independent of temperature and ionic strength, which contrasts with BCN. Much of the early work on KCN was done by Vreeman and co-workers.46−49 Thurn et al.31 measured SANS spectra and concluded that KCN formed micellar structures just like BCN, but these probably associated through (disulfide) bonding at the hydrophilic N-terminus. De Kruif and May50 measured SANS spectra of KCN micelles in which the disulfide bonds were reduced with dithiothreitol. They found structures with a dense core and a fluffy corona with a radius of 12−15 nm depending on the measurement method. Later, De Kruif et al.37 did further measurements using SANS confirming the earlier data and showed, using NMR, that BCN micelles are less dense and more dynamic than KCN micelles. Ossowski et al. report a 10484

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temperature independent association number of 30−35.39 Hence, KCN appears to be a micelle with a dense core and a (very) fluffy corona. From a structural point of view, both micelles appear to be very similar. Often, micelles, such as those from BCN, are used to adsorb materials in the hydrophobic core. However, for positively charged solutes adsorption in the corona of the micelles is present as well. Here, we investigate the adsorption of lactotransferrin, a basic protein, into the corona of the micelles but also at temperatures where the BCN micelles are dissociated. We will use SAXS measurements to unravel the structure of the coacervates of lactotransferrin with BCN or KCN. We will analyze the SAXS spectra using an appropriate model for the system at hand and measure the scattering of both lactotransferrin and BCN (the last one also as a function of temperature).

Figure 3 shows a plot of BCN scattering at temperatures between 2 and 30 °C for BCN concentrations of 4 and 16.1 g/L. The spectra were the same for increasing or decreasing temperatures and completely reproducible. It is known that BCN forms micelles at ambient temperatures34,53 but is predominantly monomeric at 2 °C. For such behavior, one would expect an increasing low Q scattering with increasing temperature as a result of increasing particle number and size. However, the spectra show a very peculiar behavior, namely, that they are virtually unchanged at both low and high Q. How the calculated curves were obtained will be explained below. It was our aim to analyze the data in a quantitative way, so as to be able to point out major changes in the system, with an eye on what is physically reasonable given other evidence. We experimented with using a polydisperse form factor for the micelles. First, this introduces adjustable parameters, and second, we found that using a so-called generalized expression (Beaucage function54) allowed a good quantification of the data with good physical significance. The generalized expression assumes one or more structural elements (see eq 1) in the scattering objects and is then fitted to the experimental scattering spectrum. We aim to use the fitted parameters to indicate trends and changes, rather than correlating them to exact physical values. We intend to get a grasp on the formation of the complexes rather than analyzing them in all possible detail. For that, further and more detailed data will be needed. In view of the pronounced correlation, we first determined experimental “structure factors” by dividing the experimental data by the lowest temperature scattering. This gives a function (not shown) that is similar to a structure factor of an excluded particle interaction. We determined the relative increase in amplitude of the scattering spectra at a few wave vector values, in particular at low Q-values and at the maximum of the correlation peak (Figure 4). The low Q-values vary only slightly with temperature, whereas the value of S(Q = Qmax) increases with temperature. The high concentration data show a clear sigmoidal increase attributed to an increasing micellization, which is then fully consistent with SANS data of Leclerc and Calmettes,55 calorimetry data of Mikheeva and Grinberg,34 and dynamic light scattering results of O’Connell, Grinberg, and De Kruif,32 O’Connell and De Kruif,38 and De Kruif and Grinberg.53 The value of Qmax = 310 Å−1 and is independent of temperature and the same for the two concentrations. It must be remarked that there are some quantitative differences with the references mentioned. We attribute this to the fact that we did not use a 25 mM sodium phosphate buffer in this investigation. Adding salt further promotes micellization (data not shown), but we intended to investigate the binding of lactotransferrin and therefore did not add salt as this would reduce the interactions. The (excluded volume) correlation peak at Q = 310 Å−1 corresponds to a half-distance (radius) of 20 nm. This amplitude increase is to be expected when volume fraction, φ, of the micelles increases at the expense of monomers. For noninteracting particles, the ordinate would be proportional to φ. For interacting particles, the ordinate would be proportional to φ × S(Q = 0, φ), where S(Q, φ) is the structure factor of the interacting particles, assuming that the form factor P(Q) does not change. For a hard sphere or repulsive particle system, S(Q = 0, φ) decreases exponentially with increasing φ. For an attractive interaction, SAhs(Q = 0, φ) increases with increasing attraction and eventually diverges at phase separation. We therefore used an adhesive hard sphere (Ahs) structure factor with the condition that φ × SAhs(Q = 0.01 Å−1, φ) is constant. This is realized for a second

II. EXPERIMENTAL METHODS Experimental Solutions. Lactotransferrin (97% purity) was obtained from DMV International (Veghel, The Netherlands). KCN (>95% purity) was prepared according to the method of Leaver and Law.51 BCN (>95% purity) was prepared according to the method of Payens and Heremans.29 Proteins were dissolved in Milli-Q water (Millipore, MA, USA) containing 0.02% NaN3 and the pH was adjusted to 6.52−6.54. After filtering through a 0.2 μm filter, the concentration was measured by absorbance (A280 − 1.7 × A320). The A320 should be a small number as it measures background scattering. Specific absorbances at 280 nm: H2O, 0.001 cm2.mg−1; lactotransferrin, 1.27 cm2.mg−1; KCN, 0.95 cm2.mg−1; BCN, 0.46 cm2.mg−1. The concentrations were: lactotransferrin, 14.35 g/L; KCN, 14.93 g/L; BCN, 16.1 g/L. SAXS Measurements. A commercially available SAXS camera, NanoSTAR (Bruker AXS, Karlruhe, Germany) with a pinhole configuration, was modified to optimize its use for weakly scattering solution samples and is described in detail by Pedersen.52

III. RESULTS AND DISCUSSION Lactotransferrin and BCN. First we will present the SAXS results of the pure compounds. Figure 2 shows the scattering

Figure 2. Scattering spectrum of lactotransferrin. The low-Q data are from light scattering. Data were fitted to a dumbbell structure.

curve of lactotransferrin. The protein structure as presented in the Swiss Protein Data Bank28 shows a dumbbell structure. Therefore, data were fitted to a halter of two lobes and the result is the drawn line in Figure 2. The radius of each lobe is 2.5 nm and the center to center distance is found to be 11 nm. The lobe size seems reasonable, but the center to center distance is somewhat large and probably indicates the presence of dimers or small aggregates, as suggested by the SLS data (Figure 2). 10485

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Figure 3. Scattering spectra of BCN at temperatures between 2 and 30 °C. Correlation peak at intermediate wave vectors increases with temperature. (A) BCN 4 g/L. (B) BCN 16.1 g/L. The fitted spectra (drawn lines, see text) were offset by a factor of 4, for clarity.

Equation 3 is the Beaucage result.54 Thus the fitting function FF(Q, a) is the sum of an adhesive hard sphere contribution IAHS(Q, r0, a2) including a structure factor S(Q). The symbols represent the following: a is a matrix containing elements a0 to a4, where a0, a1, and a3 are amplitudes; a2 is the radius of gyration of the particles, Rg; and a4 is the polymer radius of gyration, Rgp. In view of the presence of BCN polymers, we added a Debye polymer scattering term PDebye(Q, Rgp) under the assumption that the two contributions are not correlated. For the structure factor S(Q, φ), we used the expressions of Menon et al.56 for the calculation of the structure factor of adhesive hard spheres. Volume fraction φ increased with temperature (2 to 30 °C) from 0.12 to 0.2 for the 16.1 g/L data, which corresponds to a voluminosity of 12 mL/g, a reasonable value in view of the low salt concentration.. We found a value of 3.5 mL/g in a phosphate buffer.34 Panouille et al.40 observed a divergence of the viscosity at 90 g/L (in salt buffer), which would correspond to a voluminosity of 0.63/0.09 = 7 mL/g. Sood et al.57,58 found from sedimentation experiments a value of 3 mL/g for the monomer. These voluminosity results indicate that the BCN micelles are quite fluffy objects. Here we used a voluminosity of 12 mL/g for all the experiments. For the 4 g/L data, we divided volume fractions of the 16.1 g/L data by 4. With these values, we fitted the experimental data to eq 1. Equation 3 contains 5 fit parameters: a0, the amplitude of the Guinier function; a1, the radius of gyration; a2 the amplitude of the power law function, which for a given structure should be approximately constant. In fitting the data, we found that the contribution of the power law function was very small. Actually, small negative values were also found, which is not physically realistic; therefore, we set a2 = 0. The amplitude of the Debye function is a3, and a4 is the radius of gyration of a polymer chain. P1 is the power law exponent and should be 4 in the Porod limit. We used P1 = 4, but results are rather insensitive to the precise value of P1. It appeared that a value of τ, the “adhesiveness” parameter, in the expressions of S(Q, φ) of 0.25 gave a very good fit of the data, especially the low concentration data. Setting the potential well width to 0.1 then gives a value for B2/Vhs = 0. This value was used for all calculations and the volume fraction was determined by varying the temperature. The correlation length was set to 20 nm because of the position of the correlation peak in Figure 3. Therefore, the fit parameters for the data in Figure 3 were the size (Rg) of the micelles, the radius of gyration of the

Figure 4. Relative amplitudes of the scattering spectra at low Q-values and at the maximum of the correlation peak (S(Q = Qmax)) as a function of temperature. ○, S(Q = Qmax) 16.1 g/L; □, S(Q = Qmax) 4 g/L; closed symbols experimental S(Q)-values at the three lowest Q-values.

virial coefficient B2/Vhs = −2.36, where Vhs is the hard sphere volume 4/3 π (20 nm)3. After some preliminary data fitting, it appeared that B2/Vhs = 0 gave an excellent fit of the data. It is interesting to note that the effective second viral coefficient is zero, which is to be expected for particles in equilibrium with a monomeric system. The osmotic compressibility (S(Q = 0, φ)) of the particles must be very small. Adding particles will tend to dissociate them into monomers or the increased number density will be compensated by attractive interactions and as a result the osmotic pressure does not increase. Leclerc and Calmettes55 measured second virial coefficients as a function of temperature and found slightly negative values. We fitted the data to the following expression: FF(Q , a) = ln[(a0 × IAHS(Q , a1, a 2) + a3 × PDebye(Q , a4))]

(1)

IAHS(Q , r0 , a 2) = Ihspol(Q , r0 , a 2) × S(Q )

(2)

⎡ (R g)2 ⎤ ⎥ Ihspol(Q , R g , a1) = a1 × exp⎢ −Q 2· ⎢⎣ 3 ⎥⎦ ⎡ Rg ⎢ erf Q · 6 + a 2⎢ ⎢ Q ⎢⎣

( (

3 ⎤P1

)) ⎥⎥ ⎥ ⎥⎦

(3) 10486

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radius was 15 nm.34 The volume fraction (number) of the micelles increases up to 30 °C. The micelles have a small radius of gyration and a large specific volume with a compact core and a fluffy “hairy” outside. In order to resolve the precise mass distribution in the BCN micelles, it would be required to do contrast variation using a technique such as SANS. BCN into Lactotransferrin. We titrated BCN into lactotransferrin and measured the scattering spectra at each composition. The results are presented in Figure 6 and the drawn lines are fits to the data using the Beaucage function with two “levels”. For comparison we have plotted the spectra of BCN and lactotransferrin at 25 °C as well. In Figure 7 we plot the coefficients of the fit. The data were fit to a Beaucage function with two Guinier and two power law

polymer, and the scattering amplitudes. Equation 1 gave a very good fit to the data. We evaluated the fit parameters of both samples by plotting them as a function of temperature (Figure 5). The amplitude a0

Figure 5. Plot of the fit parameters (of Figure 3, BCN) as a function of temperature. For the 4 g/L (left pane) and 16.1 g/L (right pane): 0, amplitude of the Guinier function; Δ, Rg of sphere; ●, amplitude Debye function; +, Rg Debye. The amplitude of the power law function was set to zero.

increases with temperature indicating the increased number of micelles, but the amplitude will also depend on the precise nature of the interparticle (20 nm radius) interaction. We observed that the Rg of the particle is very small (about 4 and 2 nm), but it is questionable whether this has any physical significance, as the low Q-data are multiplied by the S(Q) values. It is noted that the calculated spectra show a much more detailed variation due to the use of a monodisperse structure factor S(Q, φ). Polydispersity in size and the distribution in wavelengths of the SAXS will effectively smooth the data. From SANS measurements (in 25 mM sodium phosphate buffer) we found a Rg of 8 nm and an interparticle radius of 15 nm.37 Fitting the data with the Beaucage function only gives a Rg of 12 nm. In any case, the BCN micelles are rather fluffy and mass is concentrated in their core, as one would expect. Summarizing the results of Figures 3−5, we can say that the BCN (in low salt conditions) forms fluffy micelles with an interaction radius of 20 nm. In phosphate buffer the interaction

Figure 7. Plot of the fit parameters of eq 4 (spectra of Figure 6) as a function of the mole fraction BCN. ○, amplitude Guinier; △, Rg1 (nm); □, amplitude erf1; + amplitude second Guinier level; ▲, Rg2 (nm); ●, amplitude erf2.

regimes. The fitting function FF(Q, a) is given by eq 4, following Pignon et al.59 ⎡⎡ ⎡ ⎢⎢ 2⎤ ⎢ erf Q × ⎡ a ( ) ⎢⎢ FF(Q , a) = ln⎢⎢a0 × exp⎢− Q 2 × 1 ⎥ + a 2⎢ 3 ⎦ Q ⎢ ⎣ ⎢⎢ ⎢⎣ ⎢⎣⎢⎣

( (

⎡ ⎡ ⎢ 2⎤ ⎢ erf Q × ⎡ a ( ) + ⎢⎢a3 × exp⎢− Q 2 × 4 ⎥ + a5⎢ 3 ⎦ Q ⎢ ⎣ ⎢ ⎢⎣ ⎣

( (

a1 6

3 ⎤P1

)) ⎥⎥ ⎥ ⎥⎦

a4 6

3 ⎤P2 ⎤⎤⎤

⎥⎥⎥ ⎥⎥⎥ ⎥ ⎥⎥⎥⎥ ⎥⎦ ⎥⎥⎥⎥ ⎦⎦⎦

)) ⎥⎥

(4)

In total there are 6 fit parameters. a1 and a4 are the radii of gyration. The other parameters are amplitudes. We plot the fit parameters in Figure 7 as function of the mixing mole fraction. Only the first amplitude changes with the molar fraction of BCN, which means that the number of particles increases up to a maximum. That maximum is at a mole fraction of ∼0.55. In addition, the scattering intensity at the maximum is 46 times higher than that of lactotransferrin. Since the weight concentration is almost constant during titration (BCN is 16.1 g/L and lactotransferrin is 14.3 g/L) it means that the molar mass of the aggregate is approximately 46 × 78 kDa = 3588 kDa. If the aggregate contains n molecules, than (0.45 × n × 78 kDa) + (0.55 × n × 24 kDa) = 3588 Da and it follows that n is about 74. Hence, each complex contains 33 lactotransferrin and 41 BCN molecules. This is an interesting result, as the number of BCN monomers in a pure BCN micelle is on the order of 30 to 40.32,34,53 This result seems to suggest that lactotransferrin binds into “the hairs’’ of the BCN micelle (Figure 8 gives an artist impression).

Figure 6. Plot of the scattering spectra on titrating BCN into lactotransferrin at 25 C. For comparison we plotted the scattering spectra of BCN and lactotransferrin as well. Lines are fits through the data. Low Q scattering is maximal at equimolar composition. Fit parameters are presented in Figure 7. 10487

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Figure 8. Cartoon of the binding of lactotransferrin to BCN micelles. Lactotransferrin is depicted as a dumbbell according to protein structure in the Swiss Protein Data Bank.28 Figure 10. Scattering intensities at the 4 lowest Q-values as a function of temperature, and the values of the fit parameters from eq 4. The sample was equimolar (x ≈ 0.5) lactotransferrin and BCN. Panel A, all data; panel, B detail of panel A. ○, experimental scattering intensities at lowest Q-values; □, amplitude of Guinier fit; ◊, Rg1; □, a2, amplitude power law; ▼, a3, amplitude of Guinier 2 fit; ⧫, a4, Rg2; ■, a5, amplitude power law 2.

This is similar to the interaction depicted by Jackler et al.60 for bovine serum albumin when bound to spherical polyelectrolyte brushes composed of a polystyrene core and a poly(acrylic acid) shell. We measured the changes in scattering intensity at mole fraction x ≈ 0.5, so close to the electroneutrality point, as a function of temperature (Figure 9). The data fitting was good

Lactotransferrin into KCN. We titrated lactotransferrin into KCN and the results were very similar to those for lactotransferrin into BCN. Therefore, we will not repeat the discussion, but present the results only. The scattering spectra are given in Figure 11,

Figure 9. Scattering spectra of lactotransferrin-BCN complexes as a function of wave vector at temperatures between 5 and 30 °C. The sample was equimolar (x ≈ 0.5). A few fit curves are included. Figure 11. Scattering spectra of LF-KCN complexes and fits of eq 4. For comparison also the scattering functions of KCN and LF are presented (the lower scattering spectra).

with the exception at the lowest Q-values. Clearly the Beaucage function cannot “depress” scattering intensity at low Q due to the particle interactions which are incorporated in the structure factor. Nevertheless, it is of interest to present the fit data in Figure 9 together with the experimental low Q scattering (we averaged the 4 lowest Q-values). The averaged experimental low Q values show a clear dissociation of the micelles when going to lower temperature (Figure 10). Curve fit is a sigmoidal function. Of the 6 fit parameters of the two-level Beaucage function, we notice that the first amplitude follows the experimental data, but due to the poor fit, at low Q it is above the experimental amplitude. The Rg1 is 12 nm and larger than the value from the combined BeaucageDebye fit. It illustrates that parameters are correlated and depend on the chosen fit function. Obviously the complexes dissociate and probably into monomeric complexes, i.e., one BCN plus one lactotransferrin at lower temperatures. The micellization of BCN is promoted by the binding of the lactotransferrin as the dissociation temperature is shifted to lower temperatures after binding. For pure BCN (Figure 4), the inflection point is at 14 °C, but in the presence of lactotransferrin, the inflection point is at 9 °C (Figure 10).

and in Figure 12 we present the fit coefficients as a function of mole fraction. The scattering intensity at the max (x ≈ 0.83) is about 80/2.783 = 28.7 times higher than for lactotransferrin. So, the molar mass of the complex is 78 × 28.7 = 2242 kDa. The molar fraction of KCN in the complexes is 0.83, so there are about 5 KCN molecules per lactotransferrin molecule. It follows that 5 × n × 19 + n × 78 = 2242, which gives n = 13. Thus, we have 65 KCN molecules binding with 13 lactotransferrin proteins. The molar mass of a KCN micelle would then be 1235 kDa, compared to 840 kDa for BCN. The Rg of KCN and BCN is virtually the same at 14 nm. This implies that KCN micelles are more compact than BCN micelles. The micellization of KCN is not temperature dependent while that of BCN is, and so one would expect a stronger interaction and more compact structure within the KCN micelle. Charge Neutrality of the Complexes. We downloaded the amino acid composition of BCN and KCN from the Swiss Protein Data Bank.28 We modified the sequence in that we added 10488

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From SAXS measurements we concluded that 1.2 BCN bind 1 lactotransferrin when charge neutrality is reached. The micellization of BCN is promoted by the binding of the lactotransferrin as the dissociation temperature is shifted to lower temperatures. KCN micelles bind lactotransferrin as well and in way similar to BCN. However, KCN carries only about 4 net negative charges and therefore 5 KCN are needed to neutralize the 21 positive charges of lactotransferrin. It is not easy to imagine how the 5 KCN molecules are arranged on a relatively small protein measuring about 5 nm, even more so because KCN is arranged in micellar structures. In a study of the binding of LF to native casein micelles we found (JAFC 2013) that by increasing LF concentration from 1% to 2% w/w the ratio increased from 1 to 2. Adding higher levels may have increased the ratio further. Now, one may ask (as one of the reviewers rightfully did) what’s the use of all this? We think it is extremely relevant to see that a pair of structured and unstructured proteins behaves quite similarly to a structured protein and a (large) polelectrolyte molecule, e.g., gum Arabic, carrageenan, and DNA. Since there are many unstructured proteins in body fluids (e.g., saliva) there is a good reason to assume that these proteins bind to globular proteins as well and therewith serve protective functions, e.g., against enzymatic degradation which is often based on docking of charged groups on the protein. We are hesitant to call this a chaperone activity as no folding is involved. More generally, the complexes could be used to protect proteins during processing and drug delivery, even more so because the unstructured proteins can be cross-linked easily.

Figure 12. Fitted parameters of eq 4 as a function of composition (mol fraction KCN). ○, a0, amplitude Guinier1; △, Rg1; ▲, Rg2. Other amplitudes are small and constant.

a typical phosphorylation to the serines. In addition we added glycosylated threonil groups to KCN. Subsequently, we calculated the number of charges as a function of pH using the SCRIPPS protein calculator (Figure 13). For lactotransferrin we

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.G.(Kees) de Kruif). Notes

The authors declare no competing financial interest.

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Figure 13. Net charge of the proteins as a function of pH using the SCRIPPS protein calculator. Lactotransferrin, +. The primary sequence of BCN was modified by adding 5 phosphate groups (●). Similarly for KCN (△, primary chain) we added 1 phosphate and 5 sugar groups to KCN (○). The primary sequence contains the amino acids only.

REFERENCES

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find that at pH 6.5 there would be 21 net positive charges. For BCN there are 14 net negative charges and for KCN 0 to 6 net negative charges depending on phosphorylation and glycosylation levels. It is interesting to note that the hydrophilic N-terminus half of the BCN molecule contains all the net negative charges, while the hydrophobic half is charge-neutral. The lactotransferrin−BCN complex contained 41/34 = 1.2 BCN/lactotransferrin. So indeed the charges on BCN just compensate the surface charges on lactotransferrin. The average charge density of KCN is somewhat uncertain, but somewhere between 0 and −6 at pH 6.5. From the molecular mass calculations we find 5 KCN/lactotransferrin at charge neutrality and this suggests that KCN carries 4.2 negative charges at pH 6.5, which is a realistic value. These charges are in the C-tminus of the molecule. Summary. BCN is mainly monomeric at 2 °C and pH 6.5. On increasing temperature, micellar structures are formed, which contain 40 monomers. Addition of lactotransferrin, a strong cationic protein, leads to the formation of protein−protein complexes. At low temperature the complexes stay in solution. At higher temperature the BCN forms micellar structures and the lactotransferrin is attached to the micelle structure. 10489

dx.doi.org/10.1021/la402236f | Langmuir 2013, 29, 10483−10490

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dx.doi.org/10.1021/la402236f | Langmuir 2013, 29, 10483−10490