A simple and efficient preparative electrophoretic technique has been proposed to obtain a modified creatine kinase (CK, E.C.2.7.3.2) molecule with an increased content of 25Mg in the active site. A key point of the method is the special design of a 0.9 x 12.0 cm column for ascendent electrophoresis, packed consecutively, from the bottom to the top, with layers of 30 % PAAG (polyacrylamide grade), 25Mg2+ -containing 7.5 % PAAG, enzyme-binding ADP Sepharose and 2.2 % agarose gels, based on different tris-glycine and tris-HCl separation buffer systems. The isotope substitution process was a result of simultaneous desorption of enzyme from ADP Sepharose and electrically directed extensive flow of 25Mg2+ cations through the porous gel matrix. Greater than 8-fold 25Mg enrichment, i.e. a 10.2-86.3 % increase of 25Mg contribution to total enzyme magnesium, has been reached. The modified 25Mg-rich CK samples manifest higher (2.4-fold increase) values of specific catalytic activity when compared with intact (control) ones.
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