Classification of the Binding Modes in Bovine Serum Albumin Using Terminally Substituted Alkane Analogues

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With the fluorescence probe of 8-anilino-1-naphthalenesulfonate (ANS), the binding modes of terminally substituted alkane analogues (C_nX; X = COOH, OH, CHO, NH₃, CONH₂) to bovine serum albumin (BSA) were investigated using a competitive binding technique. The Scatchard plot of the fluorometric titration of BSA with ANS showed that the maximum binding number of ANS, n_max, was 3.81, with the binding constant, K_bnd, of 1.42 × 10⁶ mol^-1 dm³. The binding modes of C_nX to BSA were analyzed based on the fluorometric titration of the ANS and BSA mixture with C_nX. C_nCOOH completely displaced the ANS bound to BSA, whereas C_nOH and C_nCHO displaced only about 40% of the ANS bound to BSA. In contrast, C_nNH₂ and C_nCONH₂ displaced very little bound ANS. By comparing these results, we classified the binding modes of C_nX to BSA into three types. Two of them are detectable with the ANS fluorescence and the remaining one is not detectable with the fluorescence.