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.
D000818:Animals; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004305:Dose-Response Relationship, Drug; D005690:Galactose; D006602:Hexosyltransferases / Q000378:metabolism*; D006801:Humans; D007103:Immobilization; D007700:Kinetics; D007768:Lactalbumin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007788:Lactose Synthase / Q000032:analysis; D009282:Naphthalenesulfonates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits / Q000276:immunology; D012685:Sepharose; D013050:Spectrometry, Fluorescence; D014052:Toluidines / Q000494:pharmacology
D009282:Naphthalenesulfonates; D014052:Toluidines; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D012685:Sepharose; D007768:Lactalbumin; D006602:Hexosyltransferases; D007788:Lactose Synthase; D005690:Galactose