Interaction mechanism of mono-PEGylated proteins in electrostatic interaction chromatography

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEG-ylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.     

PEG chain length impacts yield of solid-phase protein PEGylation and efficiency of PEGylated protein separation by ion-exchange chromatography: Insights of mechanistic models

The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono-PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387–1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid-phase PEGylation) and the PEGylated proteins were separated on the same column. Solid-phase PEGylation inhibited the production of multi-PEGylated forms and resulted in a relatively low yield of selective mono-PEGylated form. Pore diffusion may play an important role in solid-phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.     

Adsorption equilibrium of proteins on a dye-ligand adsorbent

Summary The effects of pH and ionic strength on adsorption of lysozyme and bovine serum albumin to Blue Sepharose have been studied. Isotherms for both proteins obey the Freundlich model. Lysozyme binding involves both hydrophobic and cation exchange interactions with the adsorbent, while binding of albumin is due primarily to cation exchange. Protein properties will be discussed in relation to the binding patterns.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

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.     

Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: a simple assay for tryptophan

Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds.     

Association of fibronectin with carboxy-group-modified proteins in vitro

Treatment of human immunoglobulin G, albumin and fibronectin with water-soluble carbodi-imide at pH4.75 in the presence of glycine ethyl ester resulted in an avid binding of ¹²⁵I-labelled native fibrinectin to the modified proteins. Succinoylation, reduction and alkylation or heat-denaturation had no such effect. In affinity chromatography under physiological conditions, serum was depleted of fibronectin when run through columns of the carbodi-imide-treated proteins coupled to agarose. Fractions eluted from such columns with urea were enriched in fibronectin. The binding of radiolabelled fibronectin to the carbodi-imide-treated proteins was inhibited by unlabelled fibronectin in relatively low concentrations, but also by albumin in higher concentrations. Heat-denatured albumin inhibited at concentrations approx. 10–30 times lower than native albumin. The binding reaction had a pH optimum of 6–8. It was inhibited at high ionic strength and in the presence of urea. Anionic detergents inhibited at millimolar concentrations, but non-ionic detergents did not inhibit the binding reaction. The results were interpreted as showing that: (1) fibronectin is capable of binding to itself, to immunoglobulin G and to albumin after a reduction of the negative surface charge of these proteins, and may have a general ability to bind such modified proteins; (2) this binding can take place under physiological conditions; (3) carboxy-group-modified proteins selectively bind fibronectin from serum. This novel binding phenomenon could be important in terms of the opsonin function of circulatory fibronectin. We propose that fibronectin may recognize modified (denatured) proteins and mediate their uptake by the reticuloendothelial system.     

Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-to-protein molar grafting ratio. Results suggest grafted PEG's form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area-to-volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins. Both methods accurately predicted (3% absolute error) the viscosity radii of various PEG-proteins produced using three native proteins, alpha-lactalbumin (14.2 kDa MW), beta-lactoglobulin dimer (37.4 kDa MW), and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, K(av), for PEG-proteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins.     

Affinity of uterine luminal proteins for rat blastocysts.

The binding of 125I-labelled rat uterine luminal proteins from Day-5 pregnant rats showed higher binding affinity to blastocysts than did the binding of proteins in uterine fluid from pro-oestrous rats (Day 0), rat serum albumin (RSA) or bovine serum albumin (BSA). Apparently little uptake of proteins into cells by phagocytosis or entry into the blastocoelic cavity occurred since similar results were obtained in the presence of sodium azide or cytochalasin B. Autoradiographic studies showed that the proteins were localized on the outer surface of the blastocyst. The binding was Ca2+-dependent. Denaturation of Day-5 uterine proteins at 80 degrees C reduced the counts to the values obtained with undenatured RSA and Day-0 fluids; this residual binding was considered as non-specific. The binding of labelled Day-5 uterine proteins was substantially reduced in the presence of unlabelled Day-5 proteins but to a lesser extent in the presence of RSA or rat serum. The dissociation of the bound labelled Day-5 uterine proteins occurred most rapidly in the presence of unlabelled Day-5 proteins. However, dissociation occurred within 2 h in the presence of other macromolecules, suggesting that the binding was not strong.     

Interaction of c(60)-fullerene and carboxyfullerene with proteins: docking and binding site alignment

The unique properties of fullerenes have raised the interest of using them for biomedical applications. Within this framework, the interactions of fullerenes with proteins have been an exciting research target, yet little is known about how native proteins can bind fullerenes, and what is the nature of these interactions. Moreover, though some proteins have been shown to interact with fullerenes, up to date, no crystal structure of such complexes was obtained. Here we report docking studies aimed at examining the interactions of fullerene in two forms (C60 nonsubstituted fullerene and carboxyfullerene) with four proteins that are known to bind fullerene derivatives: HIV protease, fullerene-specific antibody, human serum albumin, and bovine serum albumin. Our work provides docking models with detailed binding pockets information, which closely match available experimental data. We further compare the predicted binding sites using a novel multiple binding site alignment method. A high similarity between the physicochemical properties and surface geometry was found for fullerene's binding sites of HIV protease and the human and bovine serum albumins.     

Studies on the different forms of material reacting with antiinsulin antibodies in the fetal and adult rat.

The nature of peak B (MW = 10-12000, proinsulin?) and PEak C (MW = 50-100000, "big big" insulin?) materials detected by the double antibody (DA) procedure in elution profiles of rat sera after Sephadex G 50 or G 100 chromatography (cf. preceding companion paper) is further investigated. Peak B is converted by mild tryptic digestion in an immunoreactive material behaving in rechromatography exactly like insulin monomer. Peak C is less easily detected by the dextran coated charcoal (DCC) method; it resists 8 M urea 37 degrees C for 1 hr, is not an artifact due to the complement system; its relative importance is very much reduced in pancreatic extracts or perifusates. Incubation of biologically active 125I labelled insulin in rat sera results in appearance of labelled material behaving on chromatography like peak C natural material, having the electrophoretic mobility of rat alpha I globulins and albumin, and resisting 8 M urea, acidic pHs and 0.5 M NaCl. Similar incubation in buffer supplemented with bovine albumin results in appearance of a labelled material having the electrophoretic mobility of beef albumin; N-ethyl-maleimide provides against this binding, which might result from (S-S)-(SH) interchanges. Rat alpha globulins and albumin (but not beef albumin) cross-react with the DA procedures; they do not react with the DCC method. Insulin bound to plasma proteins reacts with both methods. It is suggested that peak C material, as detected by the DA method in rat serum, consists both of insulin covalently bound to plasma proteins and of certain plasma proteins; the DCC method detects only bound insulin. In streptozotocin treated rats, peak C material persists after the complete disappearance of insulin and proinsulin, when detected by the (DA) procedure, but disappears when detected by the DCC procedure.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Binding interaction of a biological photosensitizer with serum albumins: a biophysical study

A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.     

Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.     

Spectroscopic determination of acetylcholine esterase–inhibitor complex: determination of conformational shifts of proteins

Changes in the absorbance spectrum of tetraphenylporphyrin sulfonate (TPPS) are observed that are unique for the proteins lysozyme, luciferase, apomyoglobin, myoglobin, gamma globulin, insulin, RNAase, phosphotransacetylase, papain, ovalbumin, bovine serum albumin (BSA), protamine sulfate, and polylysine. The absorbance spectrum of porphyrins is different for native compared with heat denatured RNAase. A unique absorbance wavelength red shift is observed with trypsin when trypsin inhibitor is present, indicating that porphyrins incorporated with proteins can detect conformational changes in the protein. The absorbance spectrum of the Soret band of TPPS undergoes bathochromic shifts upon addition of local anesthetics to acetylcholine esterase (AChE), suggesting that the absorbance spectrum of porphyrins can be used as a reporter of the presence of inhibitors of AChE by indicating conformational changes on binding of the inhibitor.     

Studies on the binding of copper to dopamine beta-monooxygenase and other proteins using the Cu2+ ion-selective electrode

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone‐ion exchange chromatography (AMC‐IEC). This new method is applied to the refolding of a widely used model protein, urea‐denatured/dithiothreitol‐reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC‐IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC‐IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL^?1, the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC‐IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC‐IEC is a high efficient and low cost protein refolding method. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010