Gel isoelectric focusing for following the successive carbamylations of amino groups in chymotrypsinogen A

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The progressive transition of CTG-A ( pI ⋍ 9.4 ) to CTG-X ( pI ⋍ 3.7 ), a previously described biologically active carbamylated derivative (1–3), is demonstrated by isoelectric focusing in polyacrylamide gels. This technique allowed the visualization of 12 carbamylated protein species, whose isoelectric point could be estimated, and also allowed the true end-point of the reaction to be determined. When the carbamylation reaction was carried to completion, the X-zymogen appeared homogeneous upon either gel electrofocusing (absence of charge isomers) or SDS electrophoresis (absence of size isomers) and had the same apparent molecular weight as the A-zymogen. On the basis of the results obtained, a prediction could be made of the conditions under which the carbamylated products would exhibit maximum solubility. These results demonstrate the high resolving power and the potential of gel isoelectric focusing for following the course and determining the end-point of reactions of proteins in which the net charge of the latter is affected.