Protein electrophoresis

Abstract

Amino acid side-chains of proteins (as well as some groups added by posttranslational modification, e.g., phosphates) confer charge characteristics. Only at the pH value represented by their isoelectric point (pI) do proteins lack charge. In fact, this charge is responsible for protein solubility in aqueous solution. When placed in an electric field of field strength E, proteins will freely move towards the electrode of opposite charge. However, they move at quite different and individual rates depending on their physical characteristics and the experimental system used (Fig. 23.1). The velocity of movement, v, of a charged molecule under these conditions depends on variables described by Eq. 1; v E q = f (1) The frictional coefficient, f, describes frictional resistance to mobility and depends on factors such as protein mass (Mr), degree of compactness, matrix porosity and buffer viscosity. The net charge, q, is determined by the number of positive and negative charges in the protein arising from charged sidechains and post-translational modifications such as deamidation, acylation, or phosphorylation. Equation 1 implies that molecules will move faster as their net charge increases, the electric field strengthens or as f decreases (a function of molecular mass/shape). Molecules of similar net charge separate due to differences in frictional coefficient whereas molecules of similar mass/shape may differ widely from each other in net charge. Consequently, electrophoresis is a high resolution technique. The electric field is established by applying a voltage, V, to a pair of electrodes separated by a distance, d (Fig. 23.1), resulting in an electrical field of strength E; E V d (2) Current is carried between the electrodes by the buffer that also maintains constant pH. The most commonly-used buffer systems in protein electrophoresis are Tris-Cl or Tris-glycine. Buffers are held in reservoirs connected to each electrode and provide a constant supply of ions to the electrophoresis system throughout the separation. Ohm's law relates V to current, I, by electrical resistance, R; V = R I (3) We might predict that increasing V would result in much faster migration of molecules due to greater current. However, large voltages result in significant power generation mainly dissipated in the form of heat. The power (in Watts) generated during electrophoresis is given by Eq. 4. W = I 2 R (4) Heat generation is undesirable because it leads to loss of resolution (convection of buffer causes mixing of separated proteins), a decrease of buffer viscosity (decreases R) and, in extreme cases, structural breakdown of thermally-labile proteins. A decrease in R means that, under conditions of constant voltage, I will increase during electrophoresis in turn leading to further heat generation (Eq. 3). In practice, constant voltage conditions are used in most electrophoresis experiments but, for certain applications, a constant power supply may be used that maintains W during the experiment allowing V to change (Eq. 4). In general, conditions are selected that are adequate to separate samples in a reasonable time-frame but that avoid extensive heating. Because E may vary widely among different experimental formats, the electrophoretic mobility, m, of a sample is defined as; m n E Combining this with Eq. 1 shows that; μ E q E f q f (6) That is, proteins migrate based on the ratio of net charge to frictional coefficient. Because f is strongly mass-dependent for classes of biopolymers of similar shape (e.g., globular proteins), differences in m approximate closely to differences in charge/mass ratio. This is an incomplete description of protein electrophoresis because it excludes possible interaction of proteins with the support medium (e.g., gels), charge suppression on the protein surface or effects of the buffer composition. Thus, protein electrophoresis is largely an empirical technique. High resolution mobility data can be obtained by comparison with standard molecules of similar charge-density and shape. However, it is not usually possible to make direct measurements (as compared to comparative measurements) of Mr or shape from electrophoretic mobilities alone due to lack of detailed information on variables involved in the process. Although most proteins behave predictably in electrophoresis, there are examples of proteins of different charge/mass comigrating or of similar charge/mass separating due to differences in their electrophoretic mobility. © 2008 Humana Press.