Rauer B, Neumann E, Widengren J, Rigler R
Faculty of Chemistry, University of Bielefeld, P.O. Box 100 131, D-33501 Bielefeld, Germany.
Biophys Chem. 1996 Jan 16;58(1-2):3-12. doi: 10.1016/0301-4622(95)00080-1.
Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B( *)) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B( *) is monophasic. The association rate coefficient of the monoliganded species AChR . B is k(ass)' = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B( *) from the monomer species AChR . B( *) . B (and AChR . B(2)( *)), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R(alpha)' --> R(alpha)'' with the exchange dissociation constants: (k(diss)')(B) = 5.5(+/-1) . 10(-5) s(-1) and (k(diss)'')(B) = 3(+/-1) . 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B( *) by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B( *) are released from the AChR . B(2)( *) species. The mechanism involves associative ternary intermediates (AChR . B( *)A, AChR . B( *)A(2) and AChR . B(2)( *)A(2)). The equilibrium constants (K(A)) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R(alpha)' and R(alpha)'', respectively, are K(A)' = 1.1 . 10(-2) M and k(-A)' = 3 . 10(5) s(-1) and K(A)'' = 7.5 . 10(-2) M and k(-A)'' = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] 0.1 M.
荧光相关光谱法（FCS）适用于在单分子计数水平上测定具有不同平移扩散系数的缓慢相互作用分子的低浓度（10⁻⁸ M）。这项新技术被用于表征四甲基罗丹明标记的α-银环蛇毒素（B*）与电鳐电器官中去污剂溶解的烟碱型乙酰胆碱受体（AChR）的相互作用动力学。在AChR的准一级条件下，与B的复合物形成是单相的。单配体物种AChR·B的缔合速率系数在293 K（20℃）时为k(ass)' = 3.8×10³ s⁻¹。通过加入过量的未标记α-银环蛇毒素（B）引发结合的B从单体物种AChR·B*·B（和AChR·B₂*）上解离，这是双相的，表明B位点存在三态级联：R(α)→R(α)'→R(α)''，交换解离常数为：(k(diss)')(B) = 5.5(±1)×10⁻⁵ s⁻¹和(k(diss)'')(B) = 3(±1)×10⁻⁶ s⁻¹在293 K时。这些数据与在一个结合位点的两种不同可相互转化构象上配体交换的解离中间步骤一致。通过过量的神经递质乙酰胆碱（ACh）使结合的B解离是双相的。在[ACh] = 0.1 M时，两个B都从AChR·B₂物种中释放出来。该机制涉及缔合三元中间体（AChR·BA、AChR·BA₂和AChR·B₂A₂）。在三元复合物状态R(α)'和R(α)''中ACh的平衡常数（K(A)）和解离速率常数（k(-A)）分别为K(A)' = 1.1×10⁻² M和k(-A)' = 3×10⁵ s⁻¹以及K(A)'' = 7.5×10⁻² M和k(-A)'' = 2×10⁶ s⁻¹。FCS数据表明通常在[ACh] 1 mM时仅结合一个ACh分子的AChR单体物种（Mr = 290 000）在[ACh] 0.1 M时确实结合两个ACh分子，这具有生理重要性。
