ISOELECTRIC-FOCUSING AS A TOOL FOR THE INVESTIGATION OF POSTTRANSLATIONAL PROCESSING AND CHEMICAL MODIFICATIONS OF PROTEINS

It has been demonstrated that good agreement may be observed between computed and experimental isoelectric point (pI) values when proteins of known sequence are focused under denaturing conditions on immobilized pH gradient IPG slabs, at least in the pH range 4-7.5. Hence, discrepancies between expected and found in this experimental set-up may be reliably ascribed to some kind of post-transcriptional processing, or chemical modification, having taken place in the sample. This evaluation is made easier when the comparison is set between the pI of a parent molecule and that (or those) of one to several of its derivatives as resolved in a single experiment (for instance, as a spot row in two-dimensional maps); no previous knowledge is required in these cases about the amino acid composition of the primary structure. The effects on protein surface charge are discussed in this review mainly for two biologically relevant processes, glycosylation and phosphorylation. Then, the pI shifts are analysed for some protein modifications that may occur naturally but can also be artefactually elicited, such as NH2 terminus blocking, deamidation and thiol redox reactions. Finally, carboxymethylation and carbamylation are used to exemplify chemical treatments often applied in connection with electrophoretic techniques and involving charged residues. Procedures to be applied in order to verify whether a given modification has occurred, and often relying on the focusing of a treated specimen, are detailed in each section. Numerical examples on model proteins are also discussed. As an important field of application of the above concepts may be genetic engineering, an exhaustive bibliographic list dealing with pI evaluation and structural assessment on recombinant proteins is included.