Prieels J P, Barel A O
Biochim Biophys Acta. 1975 Jun 26;393(2):496-504. doi: 10.1016/0005-2795(75)90077-x.
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.
固定化蛋白质的内源荧光和外源荧光被用于研究α-乳白蛋白-琼脂糖与蛋白质配体之间的相互作用。人α-乳白蛋白-琼脂糖偶联物的荧光峰发生蓝移，并在半乳糖基转移酶（A蛋白）存在下猝灭，这表明两种蛋白质之间可能形成了复合物。结合到琼脂糖上的人α-乳白蛋白的天然荧光在抗人α-乳白蛋白抗体存在下特异性猝灭。这种荧光变化似乎是由于抗体与固定化抗原的结合。此外，结合染料如2-对甲苯胺基萘-6-磺酸盐的外源荧光被用于确认抗体与α-乳白蛋白-琼脂糖之间结合的存在，并获得缔合常数的值。报道的结合常数为5.6×10⁶ M⁻¹，与通过平衡透析获得的其他数据相比具有优势。
