Study of binding of thyroxin-conjugates to the thyroxin-binding proteins

In this work we studied the effects of the molecular weight (M.W.) of thyroxin (T-4)-conjugates and the sample dilution factor on the binding potential (C) of serum T-4-binding proteins for these T-4-conjugates. We prepared six T-4-conjugates with great difference in molecular weight with proteins such as IgG, apoferritin, ferritin, transferrin, and thyroglobulin using p-benzoquinone as bifunctional reagent. The conjugates prepared were characterized in terms of their M.W., the molar ratio of T-4 to the carrier protein, and their affinity to bind with the anti-T-4 antibody. The binding potential values of serum T-4-binding proteins for the T-4-conjugates were determined, following appropriate mathematical interpretation of the results, obtained through an assay system containing I-125-labeled conjugated tracers, anti-T-4 antibody in great excess compared with the concentration of the tracers, and increasing concentration of T-4-binding proteins. We conclude that the M.W. of the conjugates is a parameter which significantly influences the binding of a conjugate to the T-4-binding proteins. Additionally, for conjugates of very high M.W. (> 650,000), zero C Values were obtained using 20-fold sample dilution in the final incubation mixture.