Citrate determination with aconitase immobilized on solid support

Pig heart aconitase has been immobilized on Enzacryl AA solid support and its kinetic behaviours were studied by using a stirred bath reactor with continuous recycling. In this reactor, the best flow rate has been determined to eliminate diffusional problems. Kinetics constants, thermic stability and pH variations have been compared between the soluble and immobilized aconitase for determination of enzyme-Enzacryl AA effectivity. Stability of the soluble and immobilized aconitase was also studied after repeated use and long-time storage. While the soluble form loses its activity after 24 hr storage, the immobilized form preserves its full activity after repeated usage and long-lasting storage. Finally, an easily measurable parameter has been found to quantitate citrate. The maximum increase of absorbance, is proportional to citrate concentration in a range between 0.2 and 3.2 mM. In conclusion, these results show that the immobilized aconitase system can be used for the determination of citrate with reproductility and great sensitivity. In addition to the simplicity of the assay, great economy in enzyme consumption has been demonstrated, in contrast to the traditional methods of quantitative citrate analysis.