Side chains. Isoelectric point. Electrophoresis

RNase A is a basic protein with an isoelectric point (pi) of 9.45 and a net positive charge in neutral solution.35 However, the conversion of positively charged lysine side chains to polar, but neutrally charged, methylol adducts would be expected to lower the pi of formalin-treated RNase A. To explore this further, RNase A was treated with 5% formalin and analyzed by isoelectric focusing (IEF) gel electrophoresis. Figure 15.5a shows that the pi values were shifted into the pH 6.0-7.4 range. Figure 15.5b shows the results of IEF... 

NE is a basic protein due to the large number of arginine residues and has an isoelectric point between 10 and 11 1251. There are at least three iso-forms of NE, which can be separated by isoelectric focusing or by polyacrylamide gel electrophoresis. The istfonns have identical N-terminal sequences and similar catalvtie activity and are believed to arise from minor differences in two N-linked carbohydrate side chains. The major form contains about 22% carbohydrate. 

While all proteins contain the peptide backbone, each protein has its own characteristic sequence of side chains, which gives it its characteristic properties. Different proteins have different proportions of acidic and basic side chains, and hence have different isoelectric points, in a solution of a particular hydrogen ion concentration, some proteins move toward a cathode and others toward an anode depending upon the size of the charge as well as upon molecular size and shape, different proteins move at different speeds. This difference in behavior in an electric field is the basis of one method of separation and analysis of protein mixtures electrophoresis. 

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