Site-specifically labeled photoprotein-thyroxine conjugates using aequorinmutants containing unique cysteine residues: Applications for binding assays (part II)

The jellyfish Aequorea victoria produces a protein, aequorin, which belongs to the class of Ca2+-dependent photoproteins known for their ability to em it visible light. This property of aequorin has allowed for its as a biolum inescent label in binding assays for a variety of analytes. Due to the exce llent detection limits we demonstrated in assays for small peptides using a fusion protein between the peptide of interest and the photoprotein, our n ext goal was to expand the range of possible analytes for producing homogen eous populations of conjugates with the aequorin label to those that were n onpeptidic in nature. Recently, we prepared and characterized four aequorin mutants containing unique cysteine residues at various positions in the po lypeptide chain. In the work reported here, the four aequorin mutants were each conjugated with a maleimide-activated methyl ester derivative of thyro xine, a hormone frequently determined to evaluate thyroid function. The thy roxine-aequorin mutant conjugates were characterized in terms of the biolum inescence activities and binding properties with an anti-thyroxine monoclon al antibody for possible future employment in either heterogeneous or homog eneous binding assays for thyroxine and/or other desired analytes.