[The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins].

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T4) was prepared by N-acylation of N-(3-aminopropyl) biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T4 conjugate with one or simultaneously with two binding proteins with affinity to Bt or T4 in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T4 was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T4. The Bt-T4 conjugate also formed a specific equimolar complex with T4-binding human globulin (TBG) by the same mechanism as that for T4. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T4 conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T4. The intact T4 competitively inhibited this interaction after its addition to the system. Bt-T4 also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu(3+)-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T4 immobilized on a solid phase. Free T4 inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T4 complex was actively bound to the T4-antibody, but had practically no interaction with serum T4-binding proteins, including TBG. Probably, nonspecific interactions of the T4 residue with streptavidin in its complex with Bt-T4, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T4-binding proteins of blood serum. The Bt-T4 stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.