Interaction between Humic Acid and Lysozyme, Studied by Dynamic

Interactions of purified Aldrich humic acid (PAHA) with the protein lysozyme (LSZ) are studied with dynamic light scattering and isothermal titration ...
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Environ. Sci. Technol. 2009, 43, 591–596

Interaction between Humic Acid and Lysozyme, Studied by Dynamic Light Scattering and Isothermal Titration Calorimetry W E N F E N G T A N , * ,†,§ L U U K K . K O O P A L , † A N D W I L L E M N O R D E †,‡ Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands, and Department Biomedical Engineering, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands

Received August 26, 2008. Revised manuscript received November 20, 2008. Accepted November 24, 2008.

Interactions of purified Aldrich humic acid (PAHA) with the protein lysozyme (LSZ) are studied with dynamic light scattering and isothermal titration calorimetry by mixing LSZ and PAHA at various mass ratios. In solution LSZ is positive and PAHA is negative at the investigated pH values. Up to moderate KCl concentrations no aggregation occurs for LSZ and for PAHA aggregated particles with an average radius of 80 nm are present. Complexation of PAHA with LSZ starts as soon as PAHA is added to LSZ and is followed by aggregation when the isoelectric-point (IEP) of the complexes is approached. Aggregation is gradual for 50 mM KCl and sudden for low KCl concentrations. The aggregate size is at its maximum at the IEP of the complexes. At mass ratios beyond the IEP the aggregates partially disaggregate. Positively charged complexes of PAHA and LSZ, formed in the absence of salt, strongly aggregate upon salt addition. Mixing of LSZ and PAHA is initially enthalpically driven. Near the IEP complexation and aggregation are due to hydrophobic forces (structural reorganization) and counterion release. The observations are relevant for other HA-protein systems when the protein is positively charged.

Introduction Humic substances (HS) are among the most widely distributed organic materials in nature and their importance in agriculture and soil sciences has been acknowledged for over 150 years (1). HS possess a variety of functional groups that in natural environments are negatively charged (2, 3). In aquatic systems and soils or sediments HS act as major buffer for protons (3, 4), toxic metal ions (5-7), and organic cations, including surfactants (8). Therefore, they are also involved in bioavailabilty and transport of trace components (9, 10). Next to the binding of low molar mass components also the binding of cationic polymers is well studied, see, e.g., ref 11, but only recently the interaction between a negatively charged humic acid and a positively charged protein (lysozyme) has been investigated (12). * Corresponding author e-mail: [email protected]. † Wageningen University. § Permanent address: College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, P. R. China. ‡ University Medical Center Groningen. 10.1021/es802387u CCC: $40.75

Published on Web 12/31/2008

 2009 American Chemical Society

Proteins, especially enzymes, play an important role in biogeochemical cycles and hold potential as an indicator of soil quality in ecosystem management (13). Pathogenic functions of proteins in soils and sediments attract attention as well (14). Examples of pathogenic proteins are insecticidal protein toxins, pharmaceutical proteins (produced in transgenic plants) and infectious proteins involved in transmissible spongiform encephalopathies (causing diseases as BSE and Creutzfeldt-Jakob). Binding of proteins to other components may lead to modification of the protein structure (15) and, consequently, to a change in their biological activity (16, 17). Binding may also make proteins less susceptible to microbial degradation (18, 19) and especially in case of pathogenic proteins this could be environmentally hazardous. Proteins generally have a strong affinity to bind to soil minerals (20, 21). The study of Tan et al. (12) shows that this is also the case for HS when the HS particles and the protein are oppositely charged. Electrostatic attraction promotes complexation between HS and protein and in the iso-electric point (IEP) of the humic acid-protein complexes mutual charge compensation occurs together with some inclusion of background electrolyte ions. Complexes at the IEP form aggregates that slowly flocculate/precipitate, but there is no knowledge of the size of the aggregates that are formed at different conditions and also the role of hydrophobic interactions with the aggregation needs further study. Hydrophobic interaction can lead to structural rearrangenents, and when large aggregates are formed the proteins will become encapsulated; both of these affect the protein activity. Therefore, in this paper, size and stability of the protein-humic acid complexes are monitored as a function of the mixing ratio of the two compounds by dynamic light scattering (DLS), and the enthalpy of the interaction is investigated by isothermal titration calorimetry (ITC). By combining the information, insight is obtained in the role that hydrophobic interactions play with complexation and aggregation of protein-HS complexes. Like in our previous study (12) the well-defined compounds lysozyme (LSZ) and purified Aldrich humic acid (PAHA) are used in aqueous solutions in which the ionic strength is adjusted by potassium chloride. PAHA is not a soil HA, but its physical behavior with respect to ion binding (4, 6), surfactant binding (8) and adsorption to metal oxides (22) is similar to that of other HAs. As our previous study has shown that the physical interactions between the LSZ and HA are very important, we believe that results obtained with PAHA can be generalized. For the present purpose it is relevant to mention that comparison of cationic surfactant binding to different HAs has shown that the hydrophobicity of PAHA is relatively large (8). Knowledge of the protein-HS interaction in general, will be, in the long run, of help for the development of methods for remediation and restoration of biological activity in soils and aquifers.

Materials and Methods Purified Aldrich Humic Acid and Lysozyme. Humic acid (Aldrich H1, 675-2) is purified using the method described by Vermeer et al. (22), except for the polishing step with Dowex resin that removes the last possible traces of metal ions. After dialysis the humic acid is freeze-dried and stored in a dry desiccator. The purified product is denoted as PAHA. The elemental analysis on an ash free basis is C, 55.8%; O, 38.9%; H, 4.6%; N, 0.6% (wt), and the molar mass of PAHA is around 20 kDa (22). A stock solution of 2 g PAHA/L is made in a volumetric flask by dissolving PAHA under mild shaking VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Proton Binding to PAHA and LSZ and the Mass Ratios PAHA/LSZ to Reach the Charge Compensation Point (CCP) and the IEP (12) a

proton binding

pH 5 7.3

a

IEP titration

KCl conc. mmol/L

PAHA mmol H+/g

LSZ mass ratio mass ratio mmol at CCP at IEP + H /g (gPAHA/gLSZ)CCP (gPAHA/gLSZ)IEP

0 5 50 0 5 50

-1.85 -1.94 -2.15 -3.19 -3.31 -3.45

0.58 0.64 0.68 0.46 0.47 0.48

0.31 0.33 0.32 0.14 0.14 0.14

0.31 0.32 0.45 0.17 0.19 0.25

Negative sign indicates (net) proton dissociation.

for about 24 h at pH 10. The high pH ensures that the PAHA is well dissolved. Hen egg-white LSZ is purchased from Sigma (L-6876) and used without further purification. Its molar mass is 14.6 kDa and its point of zero charge (PZC) is at pH 10.4 (12). LSZ molecules have a nearly spherical shape and a good structural stability (23, 24). The LSZ is dissolved in water to a stock concentration of 5 g/L and stored at 4 °C. Proton binding data of PAHA and LSZ, the mass ratios (gPAHA/gLSZ)CCP to reach the mutual charge compensation point (CCP) and (gPAHA/gLSZ)IEP to reach the IEP of the complex (12) at the pH and KCl concentrations relevant for this study are summarized in Table 1. The density of negative groups of PAHA is considerably larger than the net density of positive groups of LSZ, especially at pH 7.3. The mass ratio at the CCP neglects interaction effects and is equal to the charge density (σ) ratio of the pure components: σLSZ/σPAHA. The mass ratio at the IEP of the complex is obtained with special titrations and changes in the state of protonation of the components are implicitly taken into account. A larger mass ratio at the IEP than at the CCP implies inclusion of K+ ions in the complex; the latter is substantial at 50 mM KCl (12). Water and Chemicals. Water used for the experiments is twice deionized and filtered through an activated carbon column and a micro filter (EASYpure UV); it has a resistance greater than 18.3 MΩ · cm. The inorganic chemicals used are of analytical grade quality (obtained from Merck or SigmaAldrich). Dynamic Light Scattering. Dynamic light scattering (DLS) measurements are made with an argon ion laser with a wavelength of 514.5 nm combined with a 5000 multiple τ digital correlator (AVL). All measurements are performed at a scattering angle of 90°. The laser power used is 400 mW. A refractive index matching bath of filtered cis-decalin surrounds the cylindrical scattering cell and the temperature is kept at 25 °C by means of a Haake C35 thermostat, providing an accuracy of (0.1 °C. Titrations of 8 mL solutions of 0.1 g/L LSZ with 0.1 g/L PAHA are carried out using a computercontrolled titration setup (Schott-Gera¨te) to regulate the sequential additions of titrant, the cell stirring, and the delay times. At every dosage (50 µL) the solution is stirred with a bar for 60 s, during which no scattering data are taken. Then, after a delay time of 30 s, the intensity, I, of the scattered light and the autocorrelation function are recorded. The typical number of light scattering runs per titration step is five. The pH is also recorded. Experiments were made at least in duplicate and results deviated no more than a few percent. In view of the (small) experimental errors in preparing the PAHA and LSZ solutions, the data are considered to be well reproducible. The data are analyzed by the method of cumulants (25), yielding a first moment, Γ, (decaying time) and a second moment µ2 (reflecting the polydispersity index, 592

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µ2 Γ 2). The apparent translational diffusion coefficient, D, of the scattering particles is obtained from D ) Γ ⁄ q2

(1)

where q is the scattering vector, defined as q ) 4πn ⁄ λsin(θ ⁄ 2)

(2)

where n is the refractive index of the medium (water), θ the detection angle and λ the wavelength of the incident laser beam. Assuming spherical particles the Stokes-Einstein equation is used to obtain the average hydrodynamic radius Rh Rh ) kT ⁄ 6πηD

(3)

where k is Boltzmann’s constant, T the absolute temperature, and η the viscosity of the solvent (water). The light intensity is reported as a normalized intensity, I, in order to correct for dilution. This intensity is obtained from the measured light intensity, Im, in the following way: I ) Im(V0 + Vt) ⁄ V0

(4)

where V0 is the initial volume of the solution containing the component that is titrated and Vt the volume of the solution with the titrant. Isothermal Titration Calorimetry. The heat effect upon adding a solution of PAHA to a solution of LSZ (both at the same pH and KCl concentration) is established by isothermal titration calorimetry (ITC) using a titration calorimeter (MicroCal, Northampton, NA). The calorimeter operates according to a twin principle, i.e., it determines the differential heat flow between a sample cell and a reference cell. The output signal is measured as an electric power vs time, and is integrated to evaluate the isothermal and isobaric heat effect of interaction between PAHA and LSZ. In all cases the PAHA solution is added in identical steps to the sample cell filled with 1.352 mL 0.5 g/L of LSZ solution. The reference cell contains 1.352 mL of the same solution as the sample cell except for LSZ. The concentrations of PAHA and LSZ are chosen such that mutual charge neutralization can easily occur during the titration: the total charge of PAHA in 1 mL titrant solution is always six times higher than that of LSZ in 1 mL sample solution. For each experiment there is an initial thermal equilibrium time of 1000 s, subsequently at least 25 injections of 10 µL (injection duration 25 s) are made, with a waiting time of 600 s between the injections. The stirring speed is set at 400 rpm. The titration is controlled by a PC with DigiTAM software (version 4.1.0.32, ThermoMetric AB, Sweden), which collects the data and integrates the output signal. Similarly as with the DLS measurements, duplicate experimentswerecarriedoutandshowedagoodreproducibility.

Results and Discussion Influence of Ionic Strength on Scattering Intensity and Radius of LSZ and PAHA. Prior to investigating the variations in I and Rh upon titrating LSZ with PAHA, these properties are monitored for LSZ and PAHA individually, as a function of KCl concentration, cKCl. The detailed results can be found in the Supporting Information. LSZ hardly alters its aggregation state upon addition of KCl; significant aggregation of LSZ at pH 5 sets in for cKCl > 900 mM. The hydrodynamic radius of LSZ is too small (4 nm) to be detected with our equipment. For PAHA the scattering intensity is much stronger than for LSZ but I increases only slightly and Rh is essentially constant at about 80 nm for cKCl < 400 mM and the results are very similar for pH 5 and pH 8. This indicates that also PAHA hardly alters its aggregation state at low KCl concentrations. The Rh value compares well with those observed by Vermeer for PAHA (22, 26). For cKCl > 400 mM

FIGURE 1. Scattered light intensity (A) and mean hydrodynamic radius (B) of the PAHA-LSZ particles at pH 5 in the course of LSZ titrated with PAHA. The arrows in panel A indicate the IEPs of the PAHA-LSZ complexes at 0, 5, and 50 mM KCl (see Table 1). the behavior is more complex and, depending on the pH (see the Supporting Information). Although the molar mass of PAHA is only a factor 1.4 larger than that of LSZ, the observed scattering intensity of PAHA is 30-50 times that of LSZ and the hydrodynamic radius about 20 times. Most likely this is due to the fact that PAHA forms weak aggregates that are in equilibrium with the much smaller individual molecules of a few nm. With DLS only the large aggregates can be observed as scattering is proportional to the square of the particle volume. HA monomers in equilibrium with primary HA aggregates have been observed for peat humic acid by Avena and Wilkinson (27) using fluorescence correlation spectroscopy. According to Avena and Wilkinson the rate of disaggregation of the aggregates increases strongly with increasing pH; their observations were made at an ionic strength of 5 mM. Sutton and Sposito (28) discuss the presence of primary HA aggregates in soil HA. Our results at pH 5 and 8 indicate that the primary PAHA aggregates do not change much up to about 400 mM. DLS: Interaction between PAHA and LSZ at pH 5. The interaction of LSZ and PAHA is investigated at relatively low KCl concentrations in which the hydrodynamic radius of the PAHA particles (primary aggregates) in solution is about 80 nm and that of LSZ about 4 nm. Figure 1 depicts I and Rh as a function of the PAHA/LSZ mass ratio, m-/m+, at pH 5 and three KCl concentrations. In Figure 1A it is observed that I increases from the very onset of PAHA addition to LSZ, up to about the IEP of the complex (m-/m+) 0.32 or 0.45). Beyond the IEP the changes in I are much smaller. Addition of PAHA to LSZ implies increasing the mass concentration and this adds to a higher value of I. However, at the IEP, the mass concentration has increased by a factor of 1.3-1.5 and this, by far, cannot explain the rise of I from close to zero to 80-100. Also, the I-value of the individual PAHA particles is not more than 20. Therefore, the conclusion is that, from the first addition on, negatively charged PAHA complexes with

positively charged LSZ until charge neutralization at the IEP is reached. In agreement with our foregoing paper (12) this clearly demonstrates the importance of the electrostatic attraction for the binding of PAHA to LSZ. The small changes in I at m-/m+ > (m-/m+)IEP suggest, at first sight, that further PAHA addition leaves the preformed PAHA-LSZ complexes unaffected but more detailed information can be gained from R h. The Rh data (Figure 1B) provide further insight in the mechanism of interaction between PAHA and LSZ. Before the IEP, where the rise in I (m-/m+) is more or less invariant with cKCl, Rh (m-/m+) is rather sensitive to cKCl. Initially, Rh increases more strongly for the higher ionic strengths. The increase in Rh is caused by mutual aggregation of positively charged, soluble complexes and the stability against aggregation of charged complexes is less at higher cKCl. At 50 mM KCl the aggregation is gradual which, for kinetic reasons, results in relatively small aggregates when m-/m+approaches the IEP. At low KCl concentrations sudden aggregation occurs and this leads to relatively large aggregates at the IEP. Inspecting the maxima in the curves for Rh (m-/m+) more closely reveals that they occur for no added salt and 5 mmol/L KCl at m-/m+ ) 0.32, corresponding to the IEP which, at these KCl concentrations, also corresponds to the CCP, see Table 1. At 50 mM KCl the maximum occurs around m-/m+ ) 0.45 which corresponds to the IEP, and not with the CCP. Hence, at higher ionic strength an excess of PAHA is required. This can be ascribed to the participation of K+-ions in the charge neutralization. The Rh data beyond the CCP or IEP indicate that superequivalent binding of PAHA to LSZ induces fragmentation of the aggregates. Interactions other than electrostatic ones supposedly dominate electrostatically adverse superequivalent binding. Hydrophobic interaction may be held responsible (18, 19). Disaggregation is less sensitive to the charge ratio in the complex compared to the aggregation before the IEP is reached. This complexation and aggregation behavior is very similar to those observed for a protein and an oppositely charged polysaccharide (29, 30) and for two oppositely charged block copolymers (31). DLS: Interaction between PAHA and LSZ at pH 7.2-7.6. DLS data for addition of PAHA to LSZ at pH 7.2-7.6 (initial pH 8) are given in Figure 2. The profiles of the curves for I (m-/m+) and Rh (m-/m+) are similar to those for pH 5 (Figure 1). The intensity curves indicate that complexation occurs and the Rh curves show how the aggregation proceeds. The maxima of the peaks of Rh occur for low salt concentration again at mass ratios close to the IEP ) CCP (0.15 < m-/m+ < 0.19), and for 50 mM KCl at the mass ratio of the IEP (m-/m+ ) 0.25). The maximum size of the aggregates at pH 7.2-7.6 and low ionic strength is slightly, but significantly, smaller than at pH 5. Apparently, the PAHA/LSZ mass ratio in the electrically neutral aggregate affects the size of the aggregate, such that a smaller fraction of PAHA in the aggregate at pH 7.2-7.6 yields a smaller size. At 50 mM KCl this trend is gone because in the IEP at pH 7.3 a relatively large mass of PAHA is present in the complex compared to pH 5 (pH 7.3: 67% more PAHA in IEP than in CCP; pH 5: 41% more PAHA in IEP than in CCP, see ref 12). Due to the fact that around pH 7.3 the IEP is reached at relatively low mass ratios, the total titration range covers a larger range of charge overcompensation than at pH 5. For m-/ m+ . 0.2 the values of I decrease with each subsequent addition of PAHA, suggesting partial break-up of the scattering objects. This trend is also observed in the decreasing values of Rh at higher m-/ m+. A comparison of the aggregation state before and after the IEP can be made at (approximately) equal absolute values of the charge of the complexes, i.e., the size of the aggregates at half-of (m-/m+)IEP can be compared with that at double VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Mean hydrodynamic radius (Rh) of the PAHA-LSZ particles at m-/m+ ) 0.057 and pH 5 as a function of the KCl concentration.

FIGURE 2. Scattered light intensity (A) and mean hydrodynamic radius (B) of the PAHA-LSZ particles at a final pH about 7.2-7.6 (initial pH 8) in the course of LSZ titrated with PAHA. The arrows in panel A indicate the IEPs of the PAHA-LSZ complexes at 0, 5, and 50 mM KCl (see Table 1). (m-/m+)IEP. Both for pH 7.2-7.6 (Figure 2) and for pH 5 (Figure 1) it is observed that at 0 and 0.5 mM KCl the aggregate size at double (m-/m+)IEP is larger than that at half-of (m-/m+)IEP. For 50 mM KCl the trend is opposite. These results demonstrate that it is not only the charge of the complex that determines the degree of aggregation; composition and the history of the aggregates play a role as well. This is further corroborated by experiments in which PAHA (in the cell) is titrated with LSZ, see the Supporting Information. Comparison of the results of both titrations suggests that the weakly aggregated primary PAHA particles disaggregate when LSZ is titrated with PAHA, but that this is not the case when LSZ is added to PAHA. DLS: Influence of Salt Addition to the PAHA/LSZ Complex. To investigate the stability of the PAHA-LSZ complexes before the IEP is reached, the effect of KCl addition to a complex formed at m-/m+ ) 0.057 and pH 5 (strongly positive complex) is investigated. Figure 3 displays Rh as a function of cKCl. At cKCl ) 0 Rh is very small and comparable to the value for pure PAHA. Upon adding KCl, the soluble PAHA-LSZ complexes become readily unstable and the aggregate size increases with increasing KCl concentration. At 50 mM the size of the aggregates (2500 nm) is comparable to that when the IEP is reached by complexation at a constant concentration of 50 mM KCl. A stable aggregate size of about 2800 nm occurs when 75 mM KCl is reached. It follows that the positively charged PAHA-LSZ complex is much less stable against saltinduced aggregation than either LSZ or PAHA. ITC: Interaction between PAHA and LSZ. The thermograms (see Supporting Information) of isothermal titrations of LSZ by PAHA reveal that addition of PAHA to LSZ is exothermic, i.e., heat is given off by the mixture. This indicates that the binding between the two components is energetically favorable. At constant pressure, which applies in our experi594

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FIGURE 4. Differential enthalpy of interaction between PAHA and LSZ as a function of the mass ratio PAHA-LSZ at pH 5 (top) and at initial pH 8 (bottom). The arrows indicate the IEPs of the PAHA-LSZ particles at 5 and 50 mM KCl (see Table 1). ments, the heat effects equal the changes in enthalpy associated with the PAHA-LSZ interaction. Figure 4 presents the calculated differential interaction enthalpies, ∆inth, kJ per mol PAHA (injectant). Please as a function of m-/m+ for 5 mM and 50 mM KCl and at pH 5 and pH 8 (initial). At pH 5 the solution is sufficiently buffered; at pH 8 the initial pH is lowered somewhat by the interaction but the final pH could not be measured in the cell. The trends are very similar for the two pH values and the two KCl concentrations: the first molecules of PAHA bind to the enthalpically most favorable sites on LSZ and when approaching the IEP of the complex ∆inth is essentially zero. In view of the interpretation of the light scattering results (Figures 1 and 2), the results in Figure 4 imply that formation of the (soluble) complexes is enthalpically favorable, probably because of complexation of the negative groups of PAHA with the positive groups of LSZ. This initial interaction is largely electrostatic in nature, which is corroborated by the smaller enthalpy change in the medium of higher KCl

concentration in which electrostatic interaction is more strongly screened. Then, at higher m-/m+ ratios, when the positive charge of LSZ is partly compensated by the negative PAHA charge the differential enthalpy decreases. This implies that further complexation between positive and negative groups becomes enthalpically less favorable. Near the IEP the enthalpy becomes zero and complexation and aggregation of the complexes occur without any or hardly any change in enthalpy. It follows that near the IEP complexation and aggregation of the complexes is driven by an entropy increase. When the weakly positively charged complexes aggregate, their electrostatic potentials are low and not much electrical work is done to bring them together. Both components contain hydrophobic parts and hydrophobic interaction will lead to water release and structural reorganization that both may bring the charged groups closer together. When positive and negative groups in the complexes approach each other to a distance closer than the Debye length part of their counterions are released. The release of bound water molecules and counterions from the complexes and their aggregates can well account for sufficient entropy gain to cause complexation and/or aggregation of species that are equally charged, but containing both positively and negatively charged groups. Structural rearrangements and encapsulation in aggregates may well affect the protein activity (15, 18), but for more quantitative conclusions a specific study is required. According to the DLS results, addition of PAHA beyond the IEP induces a certain degree of disaggregation (at least in 5 mM KCl solutions). Calorimetry shows that the initial disaggregation proceeds without significant enthalpy changes, implying that this trajectory of the PAHA-LSZ interaction is entropy-controlled as well. It may be hypothesized that fragmentation of the aggregates may be controlled by entropy gain of the PAHA-LSZ complexes without sacrificing too much entropy of inorganic ions when they have to serve as counterions. The conclusions of the present study that the affinity for complexation of PAHA and LSZ is high, that the complexes easily aggregate in the presence of some salt, and that (at least around the IEP of the complex) hydrophobic interaction plays a role, are expected to hold as well for other HA-cationic protein systems. Therefore, when in environmental systems both HA and cationic proteins are present complexes that easily aggregate will be formed. The aggregation is partially reversible and the extent of aggregation will be depending on pH, salt concentration, relative abundance of protein compared to HA, sequence of addition, type of HA, and type of protein. The hydrophobic interactions and encapsulation that occur with complexation and aggregation may affect the protein activity (19), but here more study is required. The interaction between the complexes and mineral surfaces may lead to adsorption phenomena that are different from those when only protein or HA is present. For instance, HA hardly adsorbs on silica, but preliminary experiments show that HA easily binds to silica covered with LSZ.

Acknowledgments We thank the WIMEK Research School of Wageningen University for providing a research grant for W-F.T. to carry out this project. W-F.T. thanks the Natural Science Foundation of China (nos. 40671088 and 40471071) for further financial support. Anton Korteweg is kindly acknowledged for his help with isothermal titration calorimetry, and Remco Fokkink is thanked for help with dynamic light scattering.

Supporting Information Available Aggregation of PAHA and LSZ upon KCl addition (Figures S1-S3), complexation and aggregation of PAHA titrated with

LSZ at pH 7.2-7.6 (reverse titration sequence as in Figure 2) (Figure S4), and the thermograms of the ITC titrations (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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(25) (26) (27) (28)

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