Isoelectric Points and Electrophoresis

Acid-Base Properties of Amino Acids 1160 24-4 Isoelectric Points and Electrophoresis 1162 24-5 Synthesis of Amino Acids 1164... 

KLOSE, J., Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals, Humangenetik, 1975, 26,231-243. 

Figure 5.1 Two-dimensional electrophoresis separates potato proteins based on their isoelectric points and molecular weights. Using a wide-scale pH gradient and large gels, 1000-2000 quantifiable proteins can be separated. The gel stained with SYPRO Rubi shows soluble tuber proteins of potato cultivar Sante.

Proteins have different isoelectric points, and in an electrochemical cell they migrate to one of the electrodes (depending on their charge, size, and shape) at different speeds. This difference in behavior is used in electrophoresis for the separation and analysis of protein mixtures. 

For isoelectric focusing (IFF) (see later section), a power supply that provides constant power is advisable. During electrophoresis, current drops significantly because of lower conductivity as carrier ampholytes focus at their isoelectric points and because of creation of zones of pure water. If a constant-voltage supply is used, frequent voltage adjustments may be necessary. Constant-current power supplies are not customarily used in lEF. Pulsed-power or pulsed-field techniques (see later section) require a power supply that can periodically change the orientation of the applied field relative to the direction of migration. 

A number of isoenzymes that are related to GST p have since been identified. A hepatic enzyme, called GST ip, has been purified in several laboratories (H27, S26), and it has been shown that this homodimeric protein has an N-terminal amino acid sequence identical to that of GST p (All, HI 6). The expression of this additional mu-class form in human liver is also subject to variation (H63). GST p and GST op are homodimers and it is now generally accepted that these two enzymes represent allelic variants encoded at the GST 1 locus identified by Board (B30) and Strange et al. (S41). Less information is available on the heterodimeric enzyme formed by the combination of p-type and ip-type subunits (H16, V4). The existence of this isoenzyme has been demonstrated by means of starch-gel electrophoresis and chromatofocusing (F2, S41) however, so far, it has not been fully characterized. A third hepatic mu-class enzyme, named GST < ), was identified by Stockman and Hayes (S36). This isoenzyme, which was present in only 1 of 20 livers examined, was shown to be immunologically related to GST p and GST ip but could be distinguished from these forms by its lower isoelectric point and the fact that, unlike GST p and GST ip, it has a blocked N terminus (P. 

The use of polyacrylamide as a gel matrix and placing it in a vertical cylinder is known as disc electrophoresis. It gets its name from the very sharp discs of compounds that are formed when made visible by staining (Chapter 29). It can be much better than horizontal electrophoresis in that in one case reported it separated over 30 components from a mixture that horizontal methods had separated only 7. The addition of a pH gradient between the electrodes can produce increased resolution, because the ions will move to where they are electrically neutral, their isoelectric point, and stay there as long as the voltage is on. This can be done either on a flat strip or in a disc, and is known as isoelectric focusing. Chapter 30. Another portion of that chapter describes the use of sodium dodecylsulfonate (SDS) to aid in the movement of neutral compounds. 

Other separations of proteins and amino acids have been achieved by pH adjustment of the solution being treated. As in electrophoresis, a molecule that is at its isoelectric point will not migrate in an electric field. However, a molecule migrates as an anion at a pH above its isoelectric point, and as a cation below its isoelectric point. Therefore, a solution of such a material at its isoelectric point can be desalted by ED with minimal loss of the material. 

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