Branched polyethylene glycol for protein precipitation

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules

Therapeutic proteins conjugated with branched poly(ethylene glycol) (PEG) have extended in vivo circulation half-lives compared to linear PEG-proteins, thought to be due partly to a greater hydrodynamic volume of branched PEG-proteins, which reduces the glomerular sieving coefficient. In this paper, viscosity radii of PEGylated alpha-lactalbumin (M(r) = 14.2 kDa) and bovine serum albumin (M(r) = 67 kDa) prepared with linear and branched PEGs (with nominal molecular weights 5, 10, 20 and 40 kDa) were compared experimentally using size exclusion chromatography (SEC). PEG adduct:protein molecular weight ratios of the PEGylated proteins covered the range 1:12 to 6:1. Direct comparisons of experimentally measured viscosity radii were found to be misleading due to differences between actual and nominal molecular weights of the PEG reagents used. Comparison with predicted viscosity radii shows that there is no significant difference between the viscosity radii of branched and linear PEG-proteins having the same total molecular weight of PEG adducts. Therefore, longer in vivo circulation half-lives of branched PEG-proteins compared to linear PEG-proteins are not explained by size difference. It is also calculated that the molecular size cut-off for glomerular filtration, 60 A for a 30 kDa PEG, matches the 30-50 A size range for the pores of the glomerular basement membrane. Finally, it is confirmed that prediction of PEG-protein viscosity radii should be based upon conservation of the total PEG adduct surface area to volume ratio for both linear and branched PEG-proteins regardless of PEGylation extent.     

Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol

This paper is focused on the local composition around a protein molecule in aqueous mixtures containing polyethylene glycol (PEG) and the solubility of proteins in water + PEG mixed solvents. Experimental data from literature regarding the preferential binding parameter were used to calculate the excesses (or deficits) of water and PEG in the vicinity of β-lactoglobulin, bovine serum albumin, lysozyme, chymotrypsinogen and ribonuclease A. It was concluded that the protein molecule is preferentially hydrated in all cases (for all proteins and PEGs investigated). The excesses of water and deficits of PEG in the vicinity of a protein molecule could be explained by a steric exclusion mechanism, i.e. the large difference in the sizes of water and PEG molecules.

The solubility of different proteins in water + PEG mixed solvent was expressed in terms of the preferential binding parameter. The slope of the logarithm of protein (lysozyme, β-lactoglobulin and bovine serum albumin) solubility versus the PEG concentration could be predicted on the basis of experimental data regarding the preferential binding parameter. For all the cases considered (various proteins, various PEGs molecular weights and various pHs), our theory predicted that PEG acts as a salting-out agent, conclusion in full agreement with experimental observations. The predicted slopes were compared with experimental values and while in some cases good agreement was found, in other cases the agreement was less satisfactory. Because the established equation is a rigorous thermodynamic one, the disagreement might occur because the experimental results used for the solubility and/or the preferential binding parameter do not correspond to thermodynamic equilibrium.     

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.     

Thermodynamic study of forces involved in bovine serum albumin and ovalbumin partitioning in aqueous two-phase systems

The partitioning of bovine serum albumin and ovalbumin in different two-phase aqueous polymer systems is investigated using a thermodynamic approach. Systems used were polyethylene glycols (PEGs) of molecular weights 1000 to 10,000 Da and Dextran T500 (500,000 Da). Ovalbumin transfer to the top phase is exothermic, which suggests an electrostatic interaction between the hydroxyl groups of PEG and the hydrophilic side chain of the protein, whereas the bovine serum albumin partition is an endothermic process that is entropically driven, which coincides with its high surface hydrophobicity. The effect of PEG molecular weight on enthalpy and heat capacity changes, associated with the partition of both proteins, is examined on the basis of a preferential interaction of low-molecular-weight PEG with the protein surface.     

Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics

Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein alpha-chymotrypsin (alpha-CT) was covalently modified with PEGs with molecular weights (M(W)) of 700, 2,000 and 5,000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (T(m)) of alpha-CT by up to 6 degrees C and the free energy of unfolding [DeltaG(U) (25 degrees C)] by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG M(W) and it leveled off after an average of four PEG molecules were bound to alpha-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG M(W). Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, PEG thermodynamically stabilizes alpha-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.     

Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides

Protein stability and solubility depend strongly on the presence of osmolytes, because of the protein preference to be solvated by either water or osmolyte. It has traditionally been assumed that only this relative preference can be measured, and that the individual solvation contributions of water and osmolyte are inaccessible. However, it is possible to determine hydration and osmolyte solvation (osmolation) separately using Kirkwood-Buff theory, and this fact has recently been utilized by several researchers. Here, we provide a thermodynamic assessment of how each surface group on proteins contributes to the overall hydration and osmolation. Our analysis is based on transfer free energy measurements with model-compounds that were previously demonstrated to allow for a very successful prediction of osmolyte-dependent protein stability. When combined with Kirkwood-Buff theory, the Transfer Model provides a space-resolved solvation pattern of the peptide unit, amino acids, and the folding/unfolding equilibrium of proteins in the presence of osmolytes. We find that the major solvation effects on protein side-chains originate from the osmolytes, and that the hydration mostly depends on the size of the side-chain. The peptide backbone unit displays a much more variable hydration in the different osmolyte solutions. Interestingly, the presence of sucrose leads to simultaneous accumulation of both the sugar and water in the vicinity of peptide groups, resulting from a saccharide accumulation that is less than the accumulation of water, a net preferential exclusion. Only the denaturing osmolyte, urea, obeys the classical solvent exchange mechanism in which the preferential interaction with the peptide unit excludes water.     

Ultrafiltration characteristics of pegylated proteins

There is growing clinical interest in the use of pegylated recombinant proteins with enhanced stability, half-life, and bioavailability. The objective of this study was to develop a quantitative understanding of the ultrafiltration characteristics of a series of pegylated proteins with different degrees of pegylation. Sieving data were compared with available theoretical models and with corresponding results for the partition coefficient in size exclusion chromatography (SEC). The sieving coefficients of the pegylated proteins depended not only on the protein size and the total molecular weight of the polyethylene glycol (PEG) but also on the number of PEG chains. This is in sharp contrast to the partition coefficient in SEC, which was uniquely determined by the total molecular weight of the PEG and protein. This difference is due to the deformation and/or elongation of the PEG chains caused by the convective flow into the membrane pores, an effect that is not present in SEC. These results provide important insights into the transport and separation characteristics of pegylated proteins.     

Relationship between the protein surface hydrophobicity and its partitioning behaviour in aqueous two-phase systems of polyethyleneglycol-dextran

In order to develop possible correlations to predict partioning behaviour of proteins, five mammalian albumins (goat, bovine, equine, human and pig ones) with similar physico-chemical properties (molecular mass and isoelectrical point) were chosen. Evaluation of the relationship between hydrophobicity and partitioning coefficient (Kr) in polyethylenglycol-dextran (PEG-DxT500) systems formed by polyethyleneglycols of different molecular mass (3350, 6000 and 10,000) was investigated by estimating relative surface hydrophobicity (So) with a fluorescent probe, 1 anilino-8-naphthalene sulfonate. No relationship between Kr and So was found for systems formed by PEG3350, while aqueous two-phase systems with PEG6000 and PEG10,000 gave better correlations. The results obtained may be explained on the basis of an increase in the interaction between the latter PEGs and the protein due to their higher hydrophobic character which increases as the PEG molecular mass does so. In this way, systems with PEGs of higher molecular mass give the highest resolution to exploit hydrophobicity in partitioning.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.     

Protein structure and hydration probed by SANS and osmotic stress

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.     

Why preferential hydration does not always stabilize the native structure of globular proteins

The observed preferential hydration of proteins in aqueous MgCl2 solutions at low pH and low salt concentration (Arakawa et al., 1990) prompted a scrutiny of possible protein stabilization by MgCl2 under the same conditions, in view of earlier observations in aqueous solutions of sugars, amino acids, and a number of salts that preferential hydration is usually accompanied by the stabilization of the native structure of globular proteins. The results of thermal transition experiments on five proteins (ribonuclease A, lysozyme, beta-lactoglobulin, chymotrypsinogen, and bovine serum albumin) revealed neither significant stabilization nor destabilization of the protein structures by MgCl2 both at acid conditions (except for ribonuclease A, which was stabilized, but to a much smaller extent than by MgSO4) and at higher pH at which MgCl2 displayed little preferential hydration. This was in contrast to the great stabilizing action of MgSO4 at the same conditions. 2-Methyl-2,4-pentanediol (MPD), which gives a very large preferential hydration of native ribonuclease A at pH 5.8 [Pittz & Timasheff (1978) Biochemistry 17, 615-623], was found to be a strong destabilizer of that protein at the same conditions. Analysis of the preferentially hydrating solvent systems led to their classification into two categories: those in which the preferential hydration is independent of solution conditions and those in which it varies with conditions. The first always stabilize protein structure, while the second do not. In the first category the predominant interaction is that of cosolvent exclusion, determined by solvent properties, with the protein being essentially inert. In the second category interactions are determined to a major extent by the chemical nature of the protein surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal solubility behavior of beta-lactoglobulin: salting-in by glycine and NaCl

The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.     

Solute exclusion from cellulose in packed columns: experimental investigation and pore volume measurements

A packed column approach was used in this investigation to determine pore volume and surface area distributions of several celluloses. Specific surface areas for Avicel PH 102, Solka Floc BW 300, and two size fractions of corn cobs that have been pretreated to remove lignin and hemicellulose were measured using this technique. In addition to measuring pore volume and specific surface area, the molecular diameters of several PEGs (polyethylene glycol) were estimated using viscosity measurements. Also, the influence of cellulose particle size, molecular diameter of PEGs, and PEG solution velocity on dispersion and tailing were investigated. Molecular diameter estimates from this investigation were 30%-35% lower than those reported in the literature. This discrepancy is due to earlier investigators using an inappropriate relationship for estimating molecular diameter from viscosity measurements. The precision of the column approach to solute exclusion was higher than that obtained by investigators using a batch approach. Dispersion increased with increasing particle size. Tailing of the elution curve was increased with increasing solute molecular diameter and elution rate. For a cellulase with a molecular diameter of 5.1 nm, estimated specific surface area ranged from 7.2 to 10.5 m(2) g(-1).     

Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior

Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon α-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Osmoelastic coupling in biological structures: formation of parallel bundles of actin filaments in a crystalline-like structure caused by osmotic stress

A high molecular weight inert molecule, poly(ethylene glycol) (PEG), or a soluble protein, ovalbumin, causes parallel bundles of actin filaments in a crystalline-like structure under physiological conditions of ionic compositions and pH. The bundle formation depends on the molecular weight of PEG, and a larger molecular weight of PEG can make the bundle at a lower concentration. Actin bundle formation has a discrete dependence on the concentration of PEG. The light scattering following PEG-induced bundle formation increased abruptly at 4.5% (w/w) PEG 6000, while at concentrations less than or equal to 4.0% (w/w) no increase was observed. Labeling actin filaments with heavy meromyosin indicated that the polarity of the filament in the bundle is random. The PEG-induced bundle formation depends on the ionic strength of the solutions and also the concentration of the filament, showing that a higher concentration of PEG was required at lower ionic strength or a lower concentration of the filament. The results described above cannot be explained on the basis of the postulation that the direct binding of PEG molecules to the actin filaments may cause bundle formation. Alternatively, the mechanism can be explained reasonably by the theory of osmoelastic coupling based on preferential exclusion of PEG molecules from the filament surface. High molecular weight molecules such as PEG should be preferentially excluded from the region adjacent to the actin filaments (exclusion layer) by steric hindrance, thereby making imbalance of osmolarity between the bulk and the exclusion layer. This imbalance puts an osmotic stress on the actin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-protein association in polymer solutions: from dilute to semidilute to concentrated

In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor protein (BLIP) in the presence of crowding agents of varying molecular mass, from monomers (ethylene glycol, glycerol, or sucrose) to polymeric agents such as different polyethylene glycols (PEGs, 0.2-8 kDa) and Ficoll. An inverse linear relation was found between translational diffusion of the proteins and viscosity in all solutions tested, in accordance with the Stokes-Einstein (SE) relation. Conversely, no simple relation was found between either rotational diffusion rates or association rates (k(on)) and viscosity. To assess the translational diffusion-independent steps along the association pathway, we introduced a new factor, alpha, which corrects the relative change in k(on) by the relative change in solution viscosity, thus measuring the deviations of the association rates from SE behavior. We found that these deviations were related to the three regimes of polymer solutions: dilute, semidilute, and concentrated. In the dilute regime PEGs interfere with TEM-BLIP association by introducing a repulsive force due to solvophobic preferential hydration, which results in slower association than predicted by the SE relation. Crossing over from the dilute to the semidilute regime results in positive deviations from SE behavior, i.e., relatively faster association rates. These can be attributed to the depletion interaction, which results in an effective attraction between the two proteins, winning over the repulsive force. In the concentrated regime, PEGs again dramatically slow down the association between TEM and BLIP, an effect that does not depend on the physical dimensions of PEGs, but rather on their mass concentration. This is probably a manifestation of the monomer-like repulsive depletion effect known to occur in concentrated polymer solutions. As a transition from moderate to high crowding agent concentration can occur in the cellular milieu, this behavior may modulate protein association in vivo, thereby modulating biological function.