Separation and characterization of a yeast alcohol dehydrogenase conjugate with theophylline

The purpose of this work is to characterize the structural difference of conjugates in order to condition the immunoreactivity of the enzymatic tracer in the homogeneous immunoassays. Conjugates between yeast alcohol dehydrogenase (ADH) and 7-theophyllincarboxyalkyl acids were obtained by the mixed-anhydride method, and characterized with kinetic, electrophoretic and chromatographic techniques (IEF, MCC, CF). The enzyme activities were found to be inversely proportional to the substitution grade but not correlated to the number of carbon atoms of the carboxyalkyl spacer arms. Only the oxidative form (ADH2) of the enzyme was found capable of reacting with theophylline derivatives in the experimental conditions. Enzyme conjugates were resolved into three components, due to the different distribution of theophylline moieties on the surface of the protein, as confirmed by examination of the tryptic patterns of the single components.