Correlations Between the Charge of Proteins and the Number of Ionizable Groups They Incorporate:  Studies Using Protein Charge Ladders, Capillary Electrophoresis, and Debye−Hückel Theory

Carbeck, Jeffrey D.; Colton, Ian J.; Anderson, Janelle R.; Deutch, J. M.; Whitesides, George M.

doi:10.1021/ja991526q

Cited by 36 publications

(86 citation statements)

References 22 publications

(38 reference statements)

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“…We have not done these experiments, and the capability of CE to resolve these differences is yet to be established. This analysis suggests (but does not prove) that values of charge (e.g., Z 0 ) extracted from charge ladders by the method of LR 1,16 and assuming ∆Z ) -1.0 will be too high in magnitude by approximately 10%. In any event, the method does not provide a quantitative estimation of Z 0 .…”

Section: Resultsmentioning

confidence: 92%

Significance of Charge Regulation in the Analysis of Protein Charge Ladders

2003

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Analysis of protein charge ladders using capillary electrophoresis (CE) provides a method of determining charges of proteins. This method has disregarded the effect of charge compensationsa response of the protein and its environment to a change in electrostatic potential on the surface of the protein. This work examines the difference in charge, ∆Z, between the first two rungs of the ladder of bovine carbonic anhydrase II (BCAII) as a function of pH and ionic strength using CE. These data were analyzed in three ways: using models based on Hückel theory and on charge regulation, and using linear regression. These analyses were in only qualitative agreement, and the differences between them suggest that simple theoretical models for the behavior of colloidal particles cannot establish the value of ∆Z accurately in proteins. Linear regression of mobilities of the rungs of charge ladderssa method proposed in earlier workscontinues to be a computationally convenient method of estimating the charge Z 0 of native proteins, but the accuracy of this method depends on the value of ∆Z. The absolute value of ∆Z cannot presently be established accurately. In the case of BCAII, we suggest ∆Z ) -0.93 for the difference in charge between the first two rungs of the charge ladder at pH ) 8.4 and 10 mM ionic strength. An estimate of the uncertainty in this value for BCAII due to uncertainties in the values of pK a of amino acids and of the hydrodynamic radius is (0.02. Other uncertainties not considered in this analysis will make this value larger. IntroductionA protein charge ladder is a collection of derivatives of a protein generated by converting its charged groups (most commonly lysine -NH 3 + but also aspartate or glutamate -CO 2 -) into electrically neutral ones ( -NHCOCH 3 or -CO 2 CH 3 ). 1 In free solution capillary electrophoresis (CE), these modified proteins separate into distinct peaks or "rungs"; each rung contains regioisomers with approximately the same charge: that is, at least nominally, the same number of modified groups. Charge ladders provide a self-calibrating tool for estimating certain basic physical parameters of proteins such as charge, 2,3 hydrodynamic radius, 4 and electrostatic contributions to the free energy of binding of ligands 5,6 and of protein folding. 7 The simplest analysis of charge ladders assumes that the charge difference (∆Z) between the consecutive rungs of a charge ladder generated by acetylation of lysine -NH 3 + groups is a full unit of charge (∆Z ) -1). Menon and Zydney have made the point that ∆Z may have a value different from -1 if the change in charge of the -amino group on acetylation (which is, in fact, -1, provided that this group is completely protonated before acetylation) is offset by a compensating change in charge elsewhere in the protein. 8 This conceptscharge compensations is well-developed and extensively modeled in colloid chemistry. 9,10 Menon and Zydney suggested that it also applies to proteins and proposed a model to analyze the adjustment of charge of the macro...

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Section: Resultsmentioning

confidence: 92%

Significance of Charge Regulation in the Analysis of Protein Charge Ladders

2003

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“…46 In four of the five proteins, the exception being R-lactalbumin, the buffer consists of 25 mM Tris base plus 192 mM glycine at pH ) 8.4. 7 This corresponds to a salt solution that is 7.9 mM in both Tris + and gly -. For R-lactalbumin, the buffer contains 20 mM NaCl in addition to the Tris and glycine present in the other solutions.…”

Section: Resultsmentioning

confidence: 99%

“…The most common procedures involve the acylation of lysine residues with acetic anhydride 3 or the conversion of aspartic and glutamic acid residues to hydroxamic groups (amidation) with hydroxylamine. 7 In free solution capillary electrophoresis, these modified proteins separate into distinct bands, or "rungs", in which each rung consists of species with the same number of chemical modifications due to similarities in their net charges. [1][2][3][4][5][6][7] As investigators have attempted to extract more quantitative information from these charge ladder studies, such as the average charge difference, ∆Z, between successive rungs of a ladder, it has become apparent that more realistic modeling of the free solution electrophoresis of proteins is needed.…”

Section: Introductionmentioning

confidence: 99%

Electrophoresis of Protein Charge Ladders: A Comparison of Experiment with Various Continuum Primitive Models

et al. 2004

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Detailed modeling of the free solution electrophoresis of five proteins (bovine R-lactalbumin, hen egg white lysozyme, bovine superoxide dismutase, human carbonic anhydrase II, and hen ovalbumin) is carried out within the framework of the continuum primitive model. Protein crystal structures and translational diffusion constants are used to design and parametrize the models. The modeling results are compared with experimental mobilities of protein charge ladders, collections of protein derivatives where the number of charge groups is varied by partial acylation of lysine residues or by amidation of glutamic and aspartic acid residues. The simplest model considered is the Yoon and Kim model of a prolate/oblate ellipsoid with uniform surface potential, electrostatics treated at the level of the linear Poisson-Boltzmann equation, and distortion of the ion atmosphere from equilibrium (ion relaxation) ignored (Yoon, B. J.; Kim, S. J. Colloid Interface Sci. 1989, 128, 275). This model provides good agreement with experiment but only if the net absolute protein charge is low or the average absolute surface, or , potential is less than ∼25 mV. Boundary element (BE) modeling is also carried out in which detailed surface models are employed and the electrostatics are solved at the level of the nonlinear Poisson-Boltzmann equation. Ion relaxation is also included in some of the BE studies. All of the experimental mobilities are in good (human carbonic anhydrase II and hen ovalbumin) to excellent (bovine R-lactalbumin, hen egg white lysozyme, and bovine superoxide dismutase) agreement with BE modeling that includes ion relaxation. We believe that these results taken as a whole serve to confirm the ability of the continuum primitive model to predict, with quantitative accuracy, the free solution electrophoretic mobilities of proteins, provided the underlying models are sufficiently realistic. When a discrepancy occurs, it may be due to error in modeling either the protein charge or the solution conformation. The models described in this work also provide a useful approach for determining values of ∆Z, the change in net charge of proteins due to the chemical modification of charged groups. Knowledge of ∆Z is essential for the use of protein charge ladders in the quantitative description of the electrostatic properties and interactions of proteins. This paper supports the view that the continuum primitive model may be more appropriate for the modeling of electrokinetics than for electrostatics. The main challenge to the accurate predictions of electrophoretic mobilities may lie primarily in the modeling of electrostatics, not electrokinetics.

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“…It is also possible to construct a charge ladder by modifying other charged groups (e.g., Glu, Asp, Arg) on a protein. 397,635 CE separates the members of a charge ladder into distinct peaks, or rungs, based on charge ( Figure 29B) provided that the electrophoretic mobilities of the rungs are sufficiently different. The combination of protein charge ladders and CE provides a set of internally consistent data useful for quantifying certain electrostatic properties of proteins.…”

Section: Protein Charge Ladders As a Tool To Probe Electrostatic Intementioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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Correlations Between the Charge of Proteins and the Number of Ionizable Groups They Incorporate: Studies Using Protein Charge Ladders, Capillary Electrophoresis, and Debye−Hückel Theory

Cited by 36 publications

References 22 publications

Significance of Charge Regulation in the Analysis of Protein Charge Ladders

Significance of Charge Regulation in the Analysis of Protein Charge Ladders

Electrophoresis of Protein Charge Ladders: A Comparison of Experiment with Various Continuum Primitive Models

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

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