AFFINITY CAPILLARY ELECTROPHORESIS APPLIED TO THE STUDIES OF INTERACTIONS OF A MEMBER OF HEAT-SHOCK PROTEIN FAMILY WITH AN IMMUNOSUPPRESSANT

The bioaffinity of receptor-ligand interactions is investigated by det ermining the binding constant (association constant or dissociation co nstant) of the resulting complex utilizing affinity capillary electrop horesis (ACE). The ACE binding assay was established with a potent imm unosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc7 0, a constitutive or cognate member of heat shock protein 70 (Hsp70) f amily. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association c onstants for the interaction between Hsc70 protein and DSG were found to be 5.7.10(4) M(-1) in a buffer with pH 6.95 and 6.3.10(4) M(-1) in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 mu M, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. T he applicability of using coated capillaries was also evaluated for pr obing Hsc70-DSG interaction.