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 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.
Thormann
Wolfgang
W
Department of Clinical Pharmacology, University of Bern, Switzerland. wolfgang.thormann@ikp.unibe.ch
Huang
Tiemin
T
Pawliszyn
Janusz
J
Mosher
Richard A
RA
D003198:Computer Simulation; D004586:Electrophoresis; D004867:Equipment Design; D014644:Genetic Variation; D006454:Hemoglobins / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D011506:Proteins / Q000302:isolation & purification*
D006454:Hemoglobins; D011506:Proteins