Relation of protein electrostatics computations to ion-exchange and electrophoretic behavior

Haggerty, L.; Lenhoff, Abraham M.

doi:10.1021/j100156a081

Cited by 42 publications

(20 citation statements)

References 2 publications

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“…Huang and Lee (1993) isolated various fluorescein conjugates of fragment B of protein A. The retention time of the conjugates correlated well with the calculated average electrostatic potential of the protein-dye conjugate based on the study of Haggerty and Lenhoff (1991).…”

Section: Introductionmentioning

confidence: 54%

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

Teske

Simon

Niebisch

et al. 2007

Biotech & Bioengineering

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Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.

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Section: Introductionmentioning

confidence: 54%

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

Teske

Simon

Niebisch

et al. 2007

Biotech & Bioengineering

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“…The procedures for calculating the average electrostatic potentials around macromolecules at specific solution conditions are discussed elsewhere. 5 The calculations were performed on a GTX 210 Silicon Graphic Workstation with the use of a 1FC2 structure data file from the protein data bank. ' The 1FC2 file describing the spatial coordinates of amino acid residues within the complex of fragment B and human IgG Fc was deposited by Deisenhofer based on his X-ray crystallographic ~t u d i e s .~ the fluorescence intensity measurements at 520 nm (excited at 495 nm) in the absence and presence of human IgG (or human IgG Fc) were then carried out by a Pandex Fluorescence Concentration Analyzer (Mundelein, IL).…”

Section: Methodsmentioning

confidence: 99%

Effect of fluorochrome location in protein A on sensing efficiency of IgG

Huang

Lee

1993

Biotech & Bioengineering

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Fragment B of protein A conjugated with fluorescein at various lysine residues is prepared and separated by using a DEAE column in anion-exchange chromatography. The binding of IgG Fc to fragment B contributes to an additional positive electric potential around fragment B. The change in the local electrostatic environment and pH can then be specifically monitored by measuring the fluorescence intensity of fluorescein conjugated with fragment B before and after the introduction of IgG. The studies for the quantitative dependence of fluorescein location on the effectiveness of fluorescein for sensing the protein A-IgG reaction are presented and discussed.

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“…The surface charge distribution, and not just the net charge, is the dominant factor that determines whether a protein adsorbs on a charged surface. In particular, "patches" of high charge on a protein surface have been postulated as being the site of interaction with chromatographic surface [33].…”

Section: Influence Of Liquid Phase Condition On a Imentioning

confidence: 99%

“…Thus, at a given pH and ionic strength, k ij can be calculated with known z p from Eqs. (31) and (33). It should be noted that z p in the above equations is the effective protein charge in the adsorption phase, which may be different from that in the bulk solution (z p.b ).…”

Section: Influence Of Liquid Phase Condition On K Ijmentioning

confidence: 99%

Analysis of statistical thermodynamic model for binary protein adsorption equilibria on cation exchange adsorbent

Xiaopeng

Sun

2007

Front. Chem. Eng. China

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knowledge of mass transfer kinetics is required to optimize the chromatographic purification process, as the process performance is mass-transfer-limited in many applications. Liquid chromatography models on the basis of continuity equations are useless unless they are fed with appropriate adsorption isotherm equations, describing the basic thermodynamic equilibria between biomolecules in the mobile phase and the stationary phase. Therefore the study of adsorption equilibrium is of major importance for liquid chromatography [1][2][3].Currently, many mathematical models have been proposed for describing the adsorption equilibrium of biomolecules. Among these models, the Langmuir-type isotherms are the most famous models and are widely used to describe the nonlinear adsorption behavior in preparative ion-exchange chromatography [4]. Expressed in the simple and explicit form, Langmuir isotherms can represent the singlecomponent adsorption equilibrium data very well. However, the Langmuir isotherms are recognized to be thermodynamically inconsistent for multi-component ion-exchange systems, and the effect of ionic strength on protein adsorption is not explicitly expressed in these models [5,6]. Based on the mass action law, the stoichiometric displacement (SD) model was proposed to study protein adsorption in ion exchange systems [1][2][3][4][5][6][7]. However, it is a non-mechanistic model on the basis of oversimplified hypotheses which restrict it to rather ideal cases. Brooks and Cramer [8] combined the SD model and the concept of macromolecule steric shielding effect together to bring forward the steric mass-action (SMA) model for ion exchange adsorption. This model is considered to be one of the best models so far for protein ion exchange adsorption equilibrium and has found many applications in multi-component ion exchange chromatography [9,10]. The SMA model has also been extended to describe the dyeligand affinity adsorption equilibrium of proteins [11]. Recently, Shen and Frey [12] further investigated the SMA model by incorporating the effects of charge regulation on the ion-exchange adsorption of proteins. Abstract A study of nonlinear competitive adsorption equilibria of proteins is of fundamental importance in understanding the behavior of preparative chromatographic separation. This work describes the nonlinear binary protein adsorption equilibria on ion exchangers by the statistical thermodynamic (ST) model. The single-component and binary protein adsorption isotherms of bovine hemoglobin (Hb) and bovine serum albumin (BSA) on SP Sepharose FF were determined by batch adsorption experiments in 0.05 mol/L sodium acetate buffer at three pH values (4.5, 5.0 and 5.5) and three NaCl concentrations (0.05, 0.10 and 0.15 mol/L) at pH 5.0. The ST model was found to depict the effects of pH and ionic strength on the single-component equilibria well, with model parameters depending on the pH and ionic strength. Moreover, the ST model gave acceptable fitting to the binary adsorption data with the fitted singlecompo...

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Relation of protein electrostatics computations to ion-exchange and electrophoretic behavior

Cited by 42 publications

References 2 publications

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

Effect of fluorochrome location in protein A on sensing efficiency of IgG

Analysis of statistical thermodynamic model for binary protein adsorption equilibria on cation exchange adsorbent

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