Metal ions and complexes as modulators of protein-interfacial electron transport at graphite electrodes

An extensive investigation of the direct (unmediated) electrochemical activity of various redox proteins at pyrolytic graphite electrodes has been undertaken. With the exception of the “blue” copper protein azurin, a profound preference for the hydrophilic “edge” over the hydrophobic “basal” plane orientation of the graphite surface is observed. This may be identified with the presence of various oxidised (CO) functionalities at the polished “edge” surface which, most probably in a random manner, constitute reversible and productive binding domains for the proteins. Conditions under which the rates and reversibility of heterogenous electron transport may be optimised depend upon the protein under examination. Well-behaved electrochemistry, indicate of diffusion-dominated heterogeneous electron transport, is modulated by electrode surface protonation (pK = 5.6) and levels of redox-inert multivalent cations, including Mg²⁺ and Cr(NH₃)³⁺₆. The electrochemistry of several proteins which have negatively charged interaction domains, including plastocyanin, and chloroplast and bacterial ferrodoxins, is promoted and stabilised by electrode surface protonation, and interfacial binding of multivalent cations which is attenuated at high ionic strength. Coversely, the electrochemistry of horse-heart cytochrome c, for which the region around the exposed heme edge carries a net positive charge, is inhibited by electrode surface protonation and destablished by the presence of multivalent cations. These patterns of behaviour may be rationalised in terms of a heterogeneous electrode surface which comprises regions of hydrophilic polar groups at which proteins may associate reversibly if resultant coulombic interactions are favourable, and regions of extensive hydrophobicity at which less reversible and (probably) degradative adsorption occurs. Within this basic model, there is considerable scope for domain selectivity which may arise from variations in medium and short range order and distribution of CO functionalities. Implications for the control of in vivo electron-transport processes are discussed.     

Ultrafiltration membranes prepared from crystalline bacterial cell surface layers as model systems for studying the influence of surface properties on protein adsorption

Crystalline bacterial cell surface layers (S-layers) were used for the preparation of the active filtration layer of ultrafiltration membranes (S-layer ultrafiltration membranes; SUMs). Since the S-layer is uniform in its pore size and morphology and its functional groups are aligned in well-defined positions, the SUMs provide ideal model systems for studying protein adsorption and membrane fouling. Due to the presence of surface-located carboxyl groups the standard SUMs have the net negative charge but exhibit basically a hydrophobic character. In order to change the net charge, the charge density and the accessibility of charged groups of the SUMs as well as their hydrophobicity, free carboxyl groups of the S-layer protein were modified with selected low molecular weight nucleophiles under conditions of preserving the crystalline lattice structure. SUMs with 1.6 to 7 charged or functional groups exposed per nm² of the membrane area were used for adsorption experiments. After solutions of differently sized and charged test proteins were filtered, the relative flux losses of distilled particle free water were measured. The results showed that the adsorption capacity of the SUMs increased with the extent of their hydrophobicity. Test proteins showed their own specific adsorption characteristics, which clearly demonstrated the difficulties in determining parameters controlling the membrane fouling. Independent of the net charge of the test proteins and that of the SUMs, the flux loss of SUMs increased with the increased charge density and an improved accessibility of the charged groups on the S-layer surface. No essential differences in the adsorption characteristics were observed between the zwitterionic SUMs of slightly surplus of free carboxyl groups and the standard SUMs of net negative charge.     

Hydrophobic and electrostatic forces control the retention of membrane peptides and proteins with an immobilised phosphatidic acid column

The retention behaviour of four membrane-associated peptides and proteins with an immobilized phosphatidic acid (PA) stationary phase was evaluated. The solutes included the cytolytic peptides gramicidin A and melittin, the integral membrane protein bacteriorhodpsin and cytochrome c, a peripheral membrane protein. Gramicidin has no nett charge and exhibited normal reversed phase-like behaviour which was largely independent of mobile phase pH. In contrast, melittin, which has a positively charged C-terminal tail, exhibited reversed phase like retention at pH 5.4 and 7.4, and was not retained at pH 3 reflecting the influence of electrostatic interactions with the negatively charged phosphatidic acid ligand. Bacteriorhodpsin was eluted at high acetonitrile concentrations at pH 3 and 5.4 and cytochrome c was only eluted at pH 3. Moreover, cytochrome c eluted in the breakthrough peak between 0 and 100% acetonitrile, demonstrating the role of electrostatic interactions with the PA surface. Overall, the results demonstrate that pH can be used to optimize the fractionation and separation of membrane proteins with immobilized lipid stationary phases.     

Interactions of α-Lactalbumin and Cytochrome c with Langmuir Monolayers of Glycerophospholipids

Interaction of cytochrome c (Cyt c), α-lactalbumin III (α-La III) with Langmuir monolayers of pure and mixed glycerophospholipids was investigated using surface pressure-area (Π-A) isotherms. The general trend was that maximum interaction between protein and phospholipid is observed for mixed (1:1 molar ratio) phospholipid monolayers. Interaction between the protein and the charged phospholipid films was found to be independent of global protein charge. Our data indicate that the proteins interact with the phospholipid films by inserting themselves into the monolayer rather than anchoring to the phospholipid head groups.     

Structural changes in apolipoproteins bound to nanoparticles

Nanoparticles are widely used in the pharmaceutical and food industries, but the consequences of exposure to the human body have not been thoroughly investigated. Apolipoprotein A-I (apoAI), the major protein in high-density lipoprotein (HDL), and other lipoproteins are found in the corona around many nanoparticles, but data on protein structural and functional effects are lacking. Here we investigate the structural consequences of the adsorption of apoAI, apolipoprotein B100 (apoB100), and HDL on polystyrene nanoparticles with different surface charges. The results of circular dichroism, fluorescence spectroscopy, and limited proteolysis experiments indicate effects on both secondary and tertiary structures. Plain and negatively charged nanoparticles induce helical structure in apoAI (negative net charge) whereas positively charged nanoparticles reduce the amount of helical structure. Plain and negatively charged particles induce a small blue shift in the tryptophan fluorescence spectrum, which is not noticed with the positively charged particles. Similar results are observed with reconstituted HDL. In apoB100, both secondary and tertiary structures are perturbed by all particles. To investigate the generality of the role of surface charge, parallel experiments were performed using human serum albumin (HSA, negative net charge) and lysozyme (positive net charge). Again, the secondary structure is most affected by nanoparticles carrying an opposite surface charge relative to the protein. Nanoparticles carrying the same net charge as the protein induce only minor structural changes in lysozyme whereas a moderate change is observed for HSA. Thus, surface charge is a critical parameter for predicting structural changes in adsorbed proteins, yet the effect is specific for each protein.     

539—electrochemical behaviour of low-potential electron-transferring proteins at the mercury electrode

This paper reports on a pulse polarographic study of low-potential electron-transferring proteins at the mercury electrode ★. The proteins studied were the negatively charged iron—sulphur cluster containing ferredoxins from spinach, and from Megasphaera elsdenii, the iron-containing rubredoxin and the FMN-containing flavodoxin both from M. elsdenii. Furthermore, the positively charged, four haem-containing cytochrome c₃ from Desulfovibrio vulgaris strain Hildenborough was studied. It was observed that the electrode reaction of these proteins could be made much more efficient when a polymer or surfactant was added, with a charge opposite to the protein. The reduction efficiency of these proteins reaches an optimum when the net charge of the protein times its concentration is about equal to the same amount of opposite charges, which was added as a polymer or surfactant.     

A new approach for the study of gas-phase ion-ion reactions using electrospray ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)₃) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]ⁿ⁺ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.     

Effect of background electrolyte on the estimation of protein hydrodynamic radius and net charge through capillary zone electrophoresis

Two physicochemical models are proposed for the estimation of both hydrodynamic radius and net charge of a protein when the capillary zone electrophoretic mobility at a given protocol, the set of pK of charged amino acids, and basic data from Protein Data Bank are available. These models also provide a rationale to interpret appropriately the effects of solvent properties on protein hydrodynamic radius and net charge. To illustrate the numerical predictions of these models, experimental data of electrophoretic mobility available in the literature for well-defined protocols are used. Five proteins are considered: lysozyme, staphylococcal nuclease, human carbonic anhydrase, bovine carbonic anhydrase, and human serum albumin. Numerical predictions of protein net charges through these models compare well with the results reported in the literature, including those found asymptotically through protein charge ladder techniques. Model calculations indicate that the hydrodynamic radius is sensitive to changes of the protein net charge and hence it cannot be assumed constant in general. Also, several limitations associated with models for estimating protein net charge and hydrodynamic radius from protein structure, amino acid sequence, and experimental electrophoretic mobility are provided and discussed. These conclusions also show clear requirements for further research.     

Ion binding to a membrane with surface charge layers

A model for the potential distribution across a charged biological membrane proposed previously by us [Biophys. J., 47 (1985) 673] is extended to a case which includes the effects of binding of monovalent cations. We assume that the membrane has a surface charge layer of thickness d which is permeable to electrolyte ions and in which the membrane-fixed charged groups are distributed at a uniform density N. We also assume that each charged group can bind one monovalent cation with an equilibrium constant K. In the limit of d → 0, keeping the product Nd constant, our model gives the most commonly used model in which ion binding is considered to occur only at the membrane surface (of zero thickness). It is shown that the amount of bound cations as well as the potential distribution are found to depend strongly on d. For example, in 0.1 M 1-1 electrolyte with K = 0.8 M⁻¹, the reduction in magnitude of the surface potential at the outer surface of the surface charge layer of 10 Å thickness is about 40 ∼ 60% of that for the membrane having the surface charge layer of zero thickness, and the deviation of the amount of bound cations for the membrane of d = 10 Å from that predicted for d = 0 is 30–40%, indicating that the conventional model assuming d = 0 leads to a serious overestimation of the surface potential as well as the amount of bound cations onto the membrane.     

The electrostatic interaction between a charged sphere and an oppositely charged planar surface and its application to protein adsorption

To calculate the electrostatic interaction between a charged sphere and a charged surface under the condition of constant charge density on the two surfaces is difficult. The theory presented in this paper provides an approximate solution to this problem when the charge of the two bodies is of opposite sign. The proposed calculation model is based on a solution of the Poisson–Boltzmann (P–B) equation for two oppositely charged planar surfaces to which the approximate integration procedure developed by Deryaguin is applied. The obtained expression is rather simple and is in good agreement with retention data for a protein in ion exchange chromatography. The developed model is physically more sound than the previously developed ‘slab’ model for protein retention. Under the experimental conditions of ion exchange chromatography of proteins, the two models give comparable numerical values for the ionic strength dependence of retention.     

Electrostatic interactions between amphoteric latex particles and proteins

The electrostatic interactions between amphoteric polymethyl methacrylate latex particles and proteins with different pI values were investigated. These latex particles possess a net positive charge at low pH, but they become negatively charged at high pH. The nature and degree of interactions between these polymer particles and proteins are primarily controlled by the electrostatic characteristics of the particles and proteins under the experimental conditions. The self-promoting adsorption process from the charge neutralization of latex particles by the proteins, which have the opposite net charge to that of the particles, leads to a rapid reduction in the zeta potential of the particles (in other words colloidal stability), and so strong flocculation occurs. On the other hand, the electrostatic repulsion forces between similarly charged latex particles and the proteins retard the adsorption of protein molecules onto the surfaces of the particles. Therefore, latex particles exhibit excellent colloidal stability over a wide range of protein concentrations. A transition from net negative charge to net positive charge, and vice versa (charge reversal), was observed when the particle surface charge density was not high enough to be predominant in the protein adsorption process.     

Frontispiece: What Are We Missing by Not Measuring the Net Charge of Proteins?

The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials—including contributions from tightly bound metal, solvent, buffer, and cosolvent ions—and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pK_a. Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein—its mass—one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pK_a values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔG_z) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔG_z contribute more to the redox potential? And what about metal binding itself? When an apo-metalloprotein, bearing minimal net negative charge (e.g., Z=−2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool—the “protein charge ladder”—and used it to begin to answer these basic questions.     

Why Are Proteins Charged? Networks of Charge–Charge Interactions in Proteins Measured by Charge Ladders and Capillary Electrophoresis

Almost all proteins contain charged amino acids. While the function in catalysis or binding of individual charges in the active site can often be identified, it is less clear how to assign function to charges beyond this region. Are they necessary for solubility? For reasons other than solubility? Can manipulating these charges change the properties of proteins? A combination of capillary electrophoresis (CE) and protein charge ladders makes it possible to study the roles of charged residues on the surface of proteins outside the active site. This method involves chemical modification of those residues to generate a large number of derivatives of the protein that differ in charge. CE separates those derivatives into groups with the same number of modified charged groups. By studying the influence of charge on the properties of proteins using charge ladders, it is possible to estimate the net charge and hydrodynamic radius and to infer the role of charged residues in ligand binding and protein folding.     

High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.     

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO(2) wafers was achieved under pH 11.6, 50mM CaCl(2), and at 70 degrees C. XG-ALG films presented mean thickness of (16+/-2)nm and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAlt adsorbed irreversibly onto XG-ALG forming homogenous monolayers approximately (4+/-1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue.     

Measurement of electrostatic interactions in protein folding with the use of protein charge ladders

This paper describes a new method for the measurement of the role of interactions between charged groups on the energetics of protein folding. This method uses capillary electrophoresis (CE) and protein charge ladders (mixtures of protein derivatives that differ incrementally in number of charged groups) to measure, in a single set of electrophoresis experiments, the free energy of unfolding (DeltaG(D-N)) of alpha-lactalbumin (alpha-LA) as a function of net charge. These same data also yield the hydrodynamic radius, R(H), and net charge measured by CE, Z(CE), of the folded and denatured proteins. Alpha-LA unfolds to a compact denatured state under mildly alkaline conditions; a small increase in R(H) (11%, 2 A) coincides with a large increase in Z(CE) (71%, -4 charge units), relative to the folded state. The increase in Z(CE), in turn, predicts a large pH dependence of free energy of unfolding (-22 kJ/mol per unit increase in pH), due to differences in proton binding in the folded and denatured states. The free energy of unfolding correlates with the square of net charge of the members of the charge ladder. The differential dependence of DeltaG(D-N) on net charge for holo-alpha-LA, (partial differential) DeltaG(D-N)/(partial differential)Z = -0.14Z kJ/mol per unit of charge. This dependence of DeltaG(D-N) on net charge is a result of a net electrostatic repulsion among charge groups on the protein. These results, together with data from pH titrations, show that both the effects of electrostatic repulsion and differences in proton binding in the folded and denatured states can play an important role in the pH dependence of this protein; the relative magnitude of these effects varies with pH. The combination of charge ladders and CE is a rapid and efficient tool that measures the contributions of electrostatics to the energetics of protein folding, and the size and charge of proteins as they unfold. All this information is obtained from a single set of electrophoresis experiments.     

Excess fibrinogen adsorption to monolayers of mixed lipids

Adsorption of fibrinogen to the monolayers of mixed lipids, dipalmitoyl phosphatidyl choline (DPPC) and eicosylamine (EA) was measured at a surface pressure of 20 mN/m by an in situ surface plasmon resonance technique. Pressure–area isotherms of DPPC + EA mixtures on water and buffer subphases indicated good lipid miscibility and some contraction of the monolayers at intermediate and higher surface pressures. Surface electric potential of the DPPC + EA monolayers showed excess values for intermediate DPPC:EA ratios. Fibrinogen adsorption and its adsorption rates from a dilute solution (0.03 mg/ml) were proportional to the fraction of EA in the monolayer indicating that protein binding was primarily driven by electrostatic interactions between positive EA charges in the monolayer and a net negative protein charge. At a higher protein concentration (0.06 mg/ml) both the fibrinogen adsorbed amount and its maximum adsorption rate showed excess values relative to the pure EA for 1:1, 2:1 and 3:1 DPPC + EA monolayers. This excess adsorption could be explained, in part, by the contraction of the monolayers with intermediate DPPC:EA ratios which resulted in an excess surface electric potential.     

Dissolution of an “adsorbate” level into a band of “catalyst” levels

It is shown that, as the number of continuum levels grows, a discrete “adsorbate” level tends to dissolve into the “catalyst” quasi-continuum levels, at least for realistic values of the interaction parameter and energy spectrum (models A and B, not model C). It is suggested that coincidence of a “dissolution maximum” with the Fermi level of the catalyst might be an optimal condition for dissociative adsorption.     

Comparison between positive and negative charging of helium droplets

Helium droplets of approximately 10⁴–10⁸ atoms have been produced in free jet expansions of liquid helium through a 5 μm nozzle into vacuum. The size distributions of the positively and negatively charged droplets were measured as a function of the electron emission current. A simple model has been developed to describe the charging process and formulas for production of singly and doubly charged droplets were derived. The ratio of the ionization cross section to the geometrical cross section and its dependence on N was obtained. In the experiment single negatively and positively charged droplets were observed. Only for sizes N larger than a certain threshold size N _th ≈ 2 × 10⁵ the positively charged droplets were found to be doubly ionized. These observations are in good agreement with the assumption, that the positively charge carriers are stable “snowballs” while the negative droplets contain an excess electron located in the inside within a metastably bound “bubble”. The threshold size N _th corresponds to a simple model in which for smaller droplets a positively charged cluster of about 50 atoms is ejected.