Isoelectrical ampholyte

M. Macka and P. R. Haddad, Elution mechanism in electrostatic ion chromatography with histidine as an isoelectric ampholyte mobile phase, /. Chromatogr. A, 884, 287, 2000. 

A large part of the dissolved amino acid exists as the ampholyte (2witterion). The isoelectric point (pi) is the pH at which the net electric charge of a dissolved amino acid molecule is 2ero. p /is expressed as... 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobiHty, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current appHcation can lead to overheating of the system. 

Palusinski, OA Allgyer, TT Mosher, RA Bier, M Saville, DA, Mathematical Modeling and Computer Simulation of Isoelectric Focusing with Electrochemically Defined Ampholytes, Biophysical Chemistry 13, 193, 1981. 

Shimao, K, Mathematical Simulation of Steady State Isoelectric Eocusing of Proteins using Carrier Ampholytes, Electrophoresis 8, 14, 1987. 

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity (- - ) and pH (- -).

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

An important quantity characterizing the amino acids, peptides and proteins is the isoelectric point, which is the value of pH at which the ampholytes do not migrate in an electrical field. This occurs when... 

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 1552-1558. 

Following similar reasoning, the adsorption pattern observed for ampholytic polyelectrolytes Ocan be explained. As illustrated in Fig. 4, polyampholytes show maximum adsorption around their isoelectric point (i.e., the pH where the net charge of the polyampholyte is zero). 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

Finally, isoelectric focusing has been a useful extension of basic gel electrophoresis in protein analysis. In this technique, a series of ampholytes is placed on the slab via electrophoresis. An ampholyte is a substance whose molecule contains both acidic and basic functional groups. Solutions of different ampholytes have different pH values. Different ampholyte molecules differ in size and therefore will have varying mobilities in the electrophoresis experiment. Thus, these molecules migrate into the slab, take... 

Capillary isoelectric focusing (CIEF) is suitable for the separation of amphoteric analyses in a pH gradient. A continuous pH gradient is built up in the column by using ampholytes under a potential field. Amphoteric analyses migrate to the point where their net charge equal to zero and they form stationary and sharply focused zones. 

FIGURE 12 Application of capillary isoelectric focusing (clEF) for the determination of apparent p/ values of rMAb samples. Capillary Bio-Rad Bio-CAP XL capillary (50 pm x 24 cm) ampholyte 80% clEF Bio-Lyte Ampholyte 3-10 (2% solution with 0.5% TEMED, 0.2% HPMC) anolyte 20 mM phosphoric acid catholyte 40 mM sodium hydroxide focusing l5kV (625V/cm) for 5 min mobilization 20 kV (833V/cm) for 25 min with zwitterions (cathodic mobilizer from Bio-Rad) capillary temperature 25°C. (Reprinted from reference 40, with permission.)... 

Tests of Purity, Isoelectric focusing (lEF) in a 0.5-mm thick horizontal slab gel was performed with LKB pH 7-9 ampholyte (Ampholine 1809-136) (5). Electrophoresis was run at 10°C for 6 h at a constant voltage of 1800 V. Protein was visualized using silver stain (9) or Sigma Coomassie Brilliant Blue G-250. 

The status of the isoelectric focusing process can be followed by the current reading. When steady state is reached where no sample migration occurs anymore, the current drops to zero. After focusing, the ampholytes and solutes are mobilized again in order to pass the detector. Mobilization can be accomplished by replacing one of the solutions in the reservoirs at the capillary end with a salt (e.g., sodium chloride), or the volume in the capillary is pushed out by applying pressure. 

An advantage of CIEF is that the total length of the capillary is filled with sample and ampholyte mixture thus, larger quantities of proteins can be separated. It therefore can be advantageously used as a sample preconcentration step prior to capillary gel electrophoresis. Furthermore, CIEF is a suitable and very precise method for measuring the isoelectric point of biomolecules. 

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