A fluorimetric study of the interactions of insolubilized human alpha-lactalbumin with galactosyl transferase (A-protein) and with anti-alpha-lactalbumin antibodies.

Intrinsic as well as extrinsic fluorescence of an immobilized protein was used for the study of the interactions between alpha-lactalbumin-Sepharose and protein ligands. The fluorescence peak of the human alpha-lactalbumin-agarose conjugate was shifted to the blue and quenched in the presence of the galactosyl transferase (A-protein), indicating the probable formation of a complex between both proteins. The natural fluorescence of human alpha-lactalbumin bound to Sepharose was specifically quenched in presence of antihuman alpha-lactalbumin antibodies. This change in fluorescence appears to be due to binding of the antibodies to the immobilized antigen. Furthermore, the extrinsic fluorescence of a bound dye such as 2-p-toluidinylnaphthalene-6-sulfonate was used to confirm the existence of binding between antibodies and alpha-lactalbumin-agarose, and to obtain values for the association constant. A value of 5.6-10(+6) M(-1) for the binding constant was reported, which compares favorably with other data obtained by equilibrium dialysis.