Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes

The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH(3)Ac, Et(2)NH(2)Ac and Et(3)NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH(3), EtNH(2), Et(2)NH and Et(3)N. Charge states derived from evaluated apparent gas-phase basicities GB(app) of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H(3)O(+) ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein-complex equilibrium in aqueous solution.     

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB(app)) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GB(app) values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom.2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is proposed.     

Charged states of proteins. Reactions of doubly protonated alkyldiamines with NH(3): solvation or deprotonation. Extension of two proton cases to multiply protonated globular proteins observed in the gas phase

The apparent gas-phase basicities (GB(app)'s) of basic sites in multiply protonated molecules, such as proteins, can be approximately predicted. An approach used by Williams and co-workers was to develop an equation for a diprotonated system, NH(3)(CH(2))(7)NH(3)(2+), and then extend it with a summation of pairwise interactions to multiply protonated systems. Experimental determinations of the rates of deprotonation of NH(3)(CH(2))(7)NH(3)(2+) by a variety of bases B, in the present work, showed that GB(app) = GB(NH(3)) = 196 kcal/mol. This result is supported also by determinations of the equilibria: NH(3)(CH(2))(p)NH(3)(2+) + NH(3) = NH(3)(CH(2))(p)NH(3) x NH(3)(2+), for p = 7, 8, 10, 12. The described experimental GB(app) is 14 kcal/mol higher than the value predicted by the equation used by Williams and co-workers but in agreement with an ab initio result by Gronert. Equations based on electrostatics are developed for the two proton and multiproton systems which allow the evaluation of GB(app) of the basic sites on proteins. These are applied for the evaluation of GB(app) of the basic sites and of N(SB), the maximum number of protons that the nondenatured proteins, carbonic anhydrase (CAII), cytochrome c (CYC), and pepsin, can hold. The N(SB) values are compared with the observed charges, Z(obs)'s, when the nondenatured proteins are produced by electrospray and found in agreement with the proposal by de la Mora that Z(obs) is determined by the number of charges provided by the droplet that contains the protein, according to the charge residue model (CRM). The GB(app) values of proteins have many other applications. They can be compared with experimental measurements and are also needed for the understanding of the thermal denaturing of charged proteins and the thermal dissociation of charged protein complexes.     

Dependency of ligand free energy landscapes on charge parameters and solvent models

We performed replica-exchange molecular dynamics (REMD) simulations of six ligands to examine the dependency of their free energy landscapes on charge parameters and solvent models. Six different charge parameter sets for each ligand were first generated by RESP and AM1-BCC methods using three different conformations independently. RESP charges showed some conformational dependency. On the other hand, AM1-BCC charges did not show conformational dependency and well reproduced the overall trend of RESP charges. The free energy landscapes obtained from the REMD simulations of ligands in vacuum, Generalized-Born (GB), and TIP3P solutions were then analyzed. We found that even small charge differences can produce qualitatively different landscapes in vacuum condition, but the differences tend to be much smaller under GB and TIP3P conditions. The simulations in the GB model well reproduced the landscapes in the TIP3P model using only a fraction of the computational cost. The protein-bound ligand conformations were rarely the global minimum states, but similar conformations were found to exist in aqueous solution without proteins in regions close to the global minimum, local minimum or intermediate states.     

Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography

A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.     

Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non‐denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non‐denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF‐separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme‐ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme‐ovotransferrin complex, can be effectively separated and extracted using non‐denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.     

Calculating solvation energies by means of a fluctuating charge model combined with continuum solvent model

Continuum solvent models have shown to be very efficient for calculating solvation energy of biomolecules in solution. However, in order to produce accurate results, besides atomic radii or volumes, an appropriate set of partial charges of the molecule is needed. Here, a set of partial charges produced by a fluctuating charge model-the atom-bond electronegativity equalization method model (ABEEMσπ) fused into molecular mechanics is used to fit for the analytical continuum electrostatics model of generalized-Born calculations. Because the partial atomic charges provided by the ABEEMσπ model can well reflect the polarization effect of the solute induced by the continuum solvent in solution, accurate and rapid calculations of the solvation energies have been performed for series of compounds involving 105 small neutral molecules, twenty kinds of dipeptides and several protein fragments. The solvation energies of small neutral molecules computed with the combination of the GB model with the fluctuating charge protocol (ABEEMσπ∕GB) show remarkable agreement with the experimental results, with a correlation coefficient of 0.97, a slope of 0.95, and a bias of 0.34 kcal∕mol. Furthermore, for twenty kinds of dipeptides and several protein fragments, the results obtained from the analytical ABEEMσπ∕GB model calculations correlate well with those from ab initio and Poisson-Boltzmann calculations. The remarkable agreement between the solvation energies computed with the ABEEMσπ∕GB model and PB model provides strong motivation for the use of ABEEMσπ∕GB solvent model in the simulation of biochemical systems.     

Constant pH molecular dynamics in generalized Born implicit solvent

A new method is proposed for constant pH molecular dynamics (MD), employing generalized Born (GB) electrostatics. Protonation states are modeled with different charge sets, and titrating residues sample a Boltzmann distribution of protonation states as the simulation progresses, using Monte Carlo sampling based on GB-derived energies. The method is applied to four different crystal structures of hen egg-white lysozyme (HEWL). pK(a) predictions derived from the simulations have root-mean-square (RMS) error of 0.82 relative to experimental values. Similarity of results between the four crystal structures shows the method to be independent of starting crystal structure; this is in contrast to most electrostatics-only models. A strong correlation between conformation and protonation state is noted and quantitatively analyzed, emphasizing the importance of sampling protonation states in conjunction with dynamics.     

A model for solvated ion emission from electrospray droplets

A model is presented which shows that the energy required to emit small singly charged and large multiply charged (protein) solvated ions from electrospray droplets can be considerably lower than those predicted by earlier models. By allowing the droplet surface to distort in reaction to the emerging ion, a more nuanced picture of the ion emission mechanism appears, one that covers the range from pure ion evaporation (PIE) for small ions to what may be termed activated pseudo-Rayleigh ion release (PRIR), a mechanism that yields charge states nearly indistinguishable from the charge residue model (CRM), for large ions. Predictions based on this model are qualitatively consistent with many experimentally observed trends.     

An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.     

Buffer salt effect on pH in the interior of an anion exchange resin

The internal pH of Q Sepharose Fast Flow anion exchange resin in equilibrium with a bis-tris acetate buffer solution is investigated as a function of buffer salt concentration. Direct evidence of a resin phase pH shift is presented. At low buffer salt concentrations of 20 mM NaCl the resin phase pH is found to be as much as 1.1 pH units greater than that of the buffer phase, approaching to within 0.1 units of the buffer phase at salt concentrations greater than 250 mM. An ideal model with no adjustable parameters based on the Boltzmann distribution and the electroneutrality condition provides excellent agreement with experimental observations. The model assumes that small ions do not bind to the resin fixed charge sites and the agreement between the model predictions and observed resin internal pH suggests that strong electrolytes do not form ion pairs with the resin fixed charge sites.     

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.     

Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry 1997, 36, 12296–12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.     

The generalized Born/volume integral implicit solvent model: estimation of the free energy of hydration using London dispersion instead of atomic surface area

A new generalized Born model for estimating the free energy of hydration is presented. The new generalized Born/volume integral (GB/VI) estimates the free energy of hydration as a classical electrostatic energy plus a cavitation energy that is not based upon atomic surface area (SA) used in GB/SA hydration models but on a VI London dispersion energy estimated from quantities already calculated in the classical electrostatic energy. The (relatively few) GB/VI model parameters are fitted to experimental data, and parameterizations for two different atomic partial charge models are presented. Comparison of the calculated and experimental free energies of hydration for 560 small molecules (both neutral and charged) shows good agreement (r(2) = 0.94).     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.     

A New Model for Multiply Charged Adduct Formation Between Peptides and Anions in Electrospray Mass Spectrometry

A new model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GB_app) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that for [Glu] Fibrinopeptide B, higher GB anions dominated in adducts observed at higher negative charge states, whereas lower GB anions appeared predominately in lower charge state adducts. Singly charged adducts were only observed for lower GB anions: HSO₄^–, I^–, CF₃COO^–. Ions that have medium GBs (NO₃^–, Br^–, H₂PO₄^–) only form adducts having −2 charge states, whereas Cl^– (higher GB) can form adducts having −3 charge states. The model portends that (1) carboxylate groups are much more basic than available amino groups; (2) apparent GBs of the various carboxylate groups on peptides do not vary substantially from one another; and (3) apparent GBs of the individual carboxylate and amino sites do not behave independently. This model was developed for negative ion attachment but an analogous mechanism is also proposed for the positive ion mode wherein (1) binding of a neutral at an amino site polarizes this amino group, but hardly affects apparent GBs of other sites; (2) proton addition (charge state augmentation) at one site can decrease the instrinsic GBs of other potential protonation sites and lower their apparent GBs.     

A novel approach for speciation of airborne chromium by convective-interaction media fast-monolithic chromatography with electrothermal atomic-absorption spectrometric detection

A new analytical procedure using an anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of chromium. The separation of Cr(VI) was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic chromatographic disc. Buffer A (0.005 mol dm(-3) TRIS-HCl, pH 8.0) and buffer B (buffer A plus 3 mol dm(-3) NH4NO3) were employed in the separation procedure. The separated chromium species were determined 'off-line' by ETAAS in 0.5 cm3 fractions. The applicability of the CIM DEAE-ETAAS procedure was investigated for the determination of airborne Cr(VI) at a plasma cutting workplace. Aerosols were collected on polycarbonate membrane filters of 8 and 0.4 microm pore size (inhalable and respirable aerosols). Alkaline extraction of filters in a heated ultrasonic bath was applied to leach chromium. Good repeatability of measurement (+/-3.0%) of the alkaline extracts was obtained for Cr(VI). The LOD (3s) was found to be 0.30 microg m(-3) Cr(VI), when 0.25 m3 of air was collected on the filter. The validation of the procedure was performed by spiking filters with Cr(VI) and by the analysis of the standard reference material CRM 545, Cr(VI) in welding dust loaded on a filter. Good recoveries for spiked samples (101-102%) and good agreement between Cr(VI) found and the reported certified value for CRM 545 were obtained. The extracts were also analysed by the FPLC-ETAAS technique. Good agreement between two techniques (r2 = 0.9978) confirmed the reliability of the CIM DEAE-ETAAS procedure developed. The main advantage of the procedure lies in the speed of the chromatographic separation (chromatographic run completed in 15 min).     

Determination of boron in ceramic reference materials by prompt gamma activation analysis using focused neutron guided beam of JRR-3M

Prompt gamma activation analysis using a focused thermal neutron guided beam at JAEA JRR-3M was applied to the determination of B in ceramic certified reference materials (BAM CRM S-003 Silicon Carbide Powder and NMIJ CRM 8004-a Silicon Nitride Powder). Cl and Si were used as internal standards to obtain linear calibration curves of B. The analytical result of B in BAM CRM S-003 was in good agreement with the certified value. The relative expanded measurement uncertainties (k = 2) were 4.8% for BAM CRM S-003 and 4.9% for NMIJ CRM 8004-a.