Page 1. EMERGING BIOCHEMICAL AND BIOPHYSICAL TECHNIQUE S Series Editor: Todd M. Schuster MODERN A... more Page 1. EMERGING BIOCHEMICAL AND BIOPHYSICAL TECHNIQUE S Series Editor: Todd M. Schuster MODERN ANALYTICAL ULTRACENTRIFUGATION Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems TODD M . SCHUSTER ...
Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate me... more Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units u...
Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variet... more Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of biglycan in denaturing solvents and to define the oligomeric states of biglycan in physiologic solvents in the presence and absence of Zn2+. In 6 M guanidine chloride, biglycan is a monomer with s0(20,w) = 2.9 S and Mz = 93,100 (where Mz is z-average molecular weight). In 0.15 M NaCl, 50 mM Tris, pH 7.5, in the absence of divalent metal ions, and at concentrations above 1 mg/ml, biglycan is predominantly dimer (s0(20,w) = 4.8 S). Under these same conditions in solvent containing 5 mM Zn2+, biglycan exists predominantly as a hexamer, with s0(20,w) = 9.4 S and Mz approximately 600,000. In either case, the oligomers dissociate reversibly. In order to determine whether the glycosaminoglycan chains or the core protein was responsible for self-association, sedimentation velocity and sedimentation equilibrium studies were conducted on the isolated components. Dermatan sulfate chains prepared from biglycan, examined in both denaturing and physiologic solvents, show no significant difference in molecular weight (Mz approximately 22,000), whether or not the solvents contain Zn2+. However, biglycan core protein strongly self-associated in physiologic solvents. Thus, the self-association of biglycan appears to be mediated by the core protein and not by its glycosaminoglycan chains.
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and rol... more Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF tha...
β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absen... more β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Sedimentation velocity experiments have identified new interactions between fluorescently-labelled β-lactoglobulin and other components in milk. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. Ruminants (e.g. cows and goats) are born without circulating immunoglobulins, which they must obtain from their mothers' milk, whilst humans obtain immunoglobulins both through milk and during gestation via the placenta. We propose that β-lactoglobulin serves to pr...
Publisher Summary This chapter discusses methods for detecting contamination of a protein sample ... more Publisher Summary This chapter discusses methods for detecting contamination of a protein sample by other proteins. Methods that provide molar quantitation of amino acids, specific prosthetic groups, or of active sites may be used to assess the purity of a sample. In cases where the specific activity of the pure material is known, it may be appropriate to determine the unit activity as a measure of relative purity. The purity is then expressed as ratio of the amount measured to the amount expected. Good quantitation has been achieved using end-group analysis and quantitative analysis for specific prosthetic groups and for enzymatic activity. Electrophoretic methods provide some of the simplest, least expensive, and the most sensitive approaches to determine the number of protein components in a sample. Depending on the type of detection method available, nanogram to microgram quantities of sample are required. Because a contaminant constitutes a fixed weight fraction of a given sample, while the detection of a contaminant depends on the total mass of contaminant, the more sample applied to a gel, the better are the chances of detecting the contaminant.
The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique ... more The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm) as a function of the experimentally determined Debye–Hückel–Henry surface charge (ZDHH) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.
It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in... more It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: 1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; 2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; 3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: 1) the intact IgG charges ranged from 0 to -13; 2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; 3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; 4...
Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protei... more Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K d, 4 mM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O 2. However, oxidation occurs rapidly and completely with H 2O 2 through a non-enzymatic process with a stoichiometry of two Fe(II)/H 2O 2, indicating complete reduction of H2O2 to H2O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemis...
In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or... more In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S translation complex that contains the 60S ribosome, mRNA, and the closed-loop factors. Previously published data by others also indicate the presence of a 50S-60S translation complex containing these same components. We have found that the abundance of this complex increased upon translational cessation, implying formation after ribosomal dissociation. Stoichiometric analyses of the abundances of the closed-loop components in the 57S complex indicate this complex is most similar to polysomal and monosomal translation complexes at the end of translation rather than at the beginning or middle of translation. In contrast, a 39S complex containing the 40S ribosome bound to mRNA and closed-loop factors was also identified with stoichiometries most simil...
Every major biopharmaceutical company incorporates a protein crystallography unit that is central... more Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalit...
As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. Th... more As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. The design philosophy of the CFA, which is to encourage hardware and software innovation, has been published. However, the hardware and software that allow and encourage future development have not been described previously. Presented here is the CFA hardware and software architecture and the rationale for how this architecture was developed. Overall, both the hardware and software architecture is modular, allowing for updates and additions over time without the need for wholesale redevelopment. The common features needed by optical systems are contained in “stacks” of electronics for synchronizing the sources and detectors to the spinning rotor. Separate computer programs operate the stacks, the motion control, the centrifuge hardware, the experiment protocol, and each optical system. These programs communicate with one another to execute functions and to provide data and status information. Each program has a database associated with it to provide nonvolatile storage and inter-process communications. While the current implementation of the CFA uses one central computer to coordinate and operate all of the systems, the modular design includes provisions for using multiple computers should that be needed for a particular optical system.
Protein science : a publication of the Protein Society, 2011
Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusiv... more Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly h...
Charge is a fundamental property of macromolecules. However, new instruments and new methods have... more Charge is a fundamental property of macromolecules. However, new instruments and new methods have been needed to explore the role of charge in determining the structure, stability, and interactions of macromolecules. An apparatus is described here that is capable of performing equilibrium electrophoresis, electrophoretic mobility or diffusion measurements. This instrument acquires absorbance data from up to 512 positions along a quartz cell. The cell permits the establishment of an electric field along its length, while retaining macroions in the field of view. The prospects and limitations of using equilibrium electrophoresis for clinical applications are explored, particularly for characterizing macromolecular reagents. Applications are described for detecting charge heterogeneity, monitoring sample stability, and for determining the role of charge in molecular structure, stability and interactions. Because equilibrium electrophoresis provides little sample fractionation, the analysis of complex fluids requires the use of specific optical labels for discriminating components.
Page 1. EMERGING BIOCHEMICAL AND BIOPHYSICAL TECHNIQUE S Series Editor: Todd M. Schuster MODERN A... more Page 1. EMERGING BIOCHEMICAL AND BIOPHYSICAL TECHNIQUE S Series Editor: Todd M. Schuster MODERN ANALYTICAL ULTRACENTRIFUGATION Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems TODD M . SCHUSTER ...
Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate me... more Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units u...
Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variet... more Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of biglycan in denaturing solvents and to define the oligomeric states of biglycan in physiologic solvents in the presence and absence of Zn2+. In 6 M guanidine chloride, biglycan is a monomer with s0(20,w) = 2.9 S and Mz = 93,100 (where Mz is z-average molecular weight). In 0.15 M NaCl, 50 mM Tris, pH 7.5, in the absence of divalent metal ions, and at concentrations above 1 mg/ml, biglycan is predominantly dimer (s0(20,w) = 4.8 S). Under these same conditions in solvent containing 5 mM Zn2+, biglycan exists predominantly as a hexamer, with s0(20,w) = 9.4 S and Mz approximately 600,000. In either case, the oligomers dissociate reversibly. In order to determine whether the glycosaminoglycan chains or the core protein was responsible for self-association, sedimentation velocity and sedimentation equilibrium studies were conducted on the isolated components. Dermatan sulfate chains prepared from biglycan, examined in both denaturing and physiologic solvents, show no significant difference in molecular weight (Mz approximately 22,000), whether or not the solvents contain Zn2+. However, biglycan core protein strongly self-associated in physiologic solvents. Thus, the self-association of biglycan appears to be mediated by the core protein and not by its glycosaminoglycan chains.
Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and rol... more Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF tha...
β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absen... more β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Sedimentation velocity experiments have identified new interactions between fluorescently-labelled β-lactoglobulin and other components in milk. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. Ruminants (e.g. cows and goats) are born without circulating immunoglobulins, which they must obtain from their mothers' milk, whilst humans obtain immunoglobulins both through milk and during gestation via the placenta. We propose that β-lactoglobulin serves to pr...
Publisher Summary This chapter discusses methods for detecting contamination of a protein sample ... more Publisher Summary This chapter discusses methods for detecting contamination of a protein sample by other proteins. Methods that provide molar quantitation of amino acids, specific prosthetic groups, or of active sites may be used to assess the purity of a sample. In cases where the specific activity of the pure material is known, it may be appropriate to determine the unit activity as a measure of relative purity. The purity is then expressed as ratio of the amount measured to the amount expected. Good quantitation has been achieved using end-group analysis and quantitative analysis for specific prosthetic groups and for enzymatic activity. Electrophoretic methods provide some of the simplest, least expensive, and the most sensitive approaches to determine the number of protein components in a sample. Depending on the type of detection method available, nanogram to microgram quantities of sample are required. Because a contaminant constitutes a fixed weight fraction of a given sample, while the detection of a contaminant depends on the total mass of contaminant, the more sample applied to a gel, the better are the chances of detecting the contaminant.
The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique ... more The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm) as a function of the experimentally determined Debye–Hückel–Henry surface charge (ZDHH) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.
It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in... more It has been known since the 1930’s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: 1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; 2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; 3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: 1) the intact IgG charges ranged from 0 to -13; 2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; 3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; 4...
Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protei... more Friedreich’s ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K d, 4 mM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O 2. However, oxidation occurs rapidly and completely with H 2O 2 through a non-enzymatic process with a stoichiometry of two Fe(II)/H 2O 2, indicating complete reduction of H2O2 to H2O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemis...
In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or... more In eukaryotic translation the 60S ribosome subunit has not been proposed to interact with mRNA or closed-loop factors eIF4E, eIF4G, and PAB1. Using analytical ultracentrifugation with fluorescent detection system, we have identified a 57S translation complex that contains the 60S ribosome, mRNA, and the closed-loop factors. Previously published data by others also indicate the presence of a 50S-60S translation complex containing these same components. We have found that the abundance of this complex increased upon translational cessation, implying formation after ribosomal dissociation. Stoichiometric analyses of the abundances of the closed-loop components in the 57S complex indicate this complex is most similar to polysomal and monosomal translation complexes at the end of translation rather than at the beginning or middle of translation. In contrast, a 39S complex containing the 40S ribosome bound to mRNA and closed-loop factors was also identified with stoichiometries most simil...
Every major biopharmaceutical company incorporates a protein crystallography unit that is central... more Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalit...
As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. Th... more As part of the Open AUC Project, the CFA is the next generation of analytical ultracentrifuge. The design philosophy of the CFA, which is to encourage hardware and software innovation, has been published. However, the hardware and software that allow and encourage future development have not been described previously. Presented here is the CFA hardware and software architecture and the rationale for how this architecture was developed. Overall, both the hardware and software architecture is modular, allowing for updates and additions over time without the need for wholesale redevelopment. The common features needed by optical systems are contained in “stacks” of electronics for synchronizing the sources and detectors to the spinning rotor. Separate computer programs operate the stacks, the motion control, the centrifuge hardware, the experiment protocol, and each optical system. These programs communicate with one another to execute functions and to provide data and status information. Each program has a database associated with it to provide nonvolatile storage and inter-process communications. While the current implementation of the CFA uses one central computer to coordinate and operate all of the systems, the modular design includes provisions for using multiple computers should that be needed for a particular optical system.
Protein science : a publication of the Protein Society, 2011
Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusiv... more Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly h...
Charge is a fundamental property of macromolecules. However, new instruments and new methods have... more Charge is a fundamental property of macromolecules. However, new instruments and new methods have been needed to explore the role of charge in determining the structure, stability, and interactions of macromolecules. An apparatus is described here that is capable of performing equilibrium electrophoresis, electrophoretic mobility or diffusion measurements. This instrument acquires absorbance data from up to 512 positions along a quartz cell. The cell permits the establishment of an electric field along its length, while retaining macroions in the field of view. The prospects and limitations of using equilibrium electrophoresis for clinical applications are explored, particularly for characterizing macromolecular reagents. Applications are described for detecting charge heterogeneity, monitoring sample stability, and for determining the role of charge in molecular structure, stability and interactions. Because equilibrium electrophoresis provides little sample fractionation, the analysis of complex fluids requires the use of specific optical labels for discriminating components.
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