High-resolution computer simulation of the dynamics of isoelectric focusing of proteins

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High‐resolution focusing data of four hemoglobin variants...

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Bibliographic Details
Published in:Electrophoresis 2004-01, Vol.25 (2), p.324-337
Main Authors: Thormann, Wolfgang, Huang, Tiemin, Pawliszyn, Janusz, Mosher, Richard A.
Format: Article
Language:English
Subjects:
Capillary isoelectric focusing
Computer Simulation
Electrophoresis
Equipment Design
Genetic Variation
Hemoglobin
Hemoglobins - genetics
Hemoglobins - isolation & purification
Humans
Hydrogen-Ion Concentration
Isoelectric Focusing
Kinetics
Proteins - isolation & purification
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Summary:A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High‐resolution focusing data of four hemoglobin variants in a convection‐free medium are presented for pH 3–10 and pH 5–8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5–10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well‐known double‐peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3–10 and pH 5–8 gradients are shown to qualitatively agree well with experimental data obtained by whole‐column optical imaging.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.200305749