Amperometric assay for aldolase activity: Antibody-catalyzed ferrocenylamine formation

Screening of new catalysts for aldolase activity is a major task in bioorganic chemistry. For this purpose, fast and convenient methods are required for the detection of the catalysts. We have developed the first amperometric assay for aldol or retro-aldol catalytic activity. A new ferrocenealdol derivative was synthesized with redox activity significantly different from that of ferrocenylamine. It was shown that the reaction between aldolase antibody 38C2 and a ferrocene-aldol substrate generated free ferrocenylamine, which could be detected and quantified by simple electrochemical measurement. The amperometric assay was applied to perform a Michaelis-Menten analysis of catalytic antibody 38C2 in order to determine the enzymatic kinetic parameters.