Iio T, Kondo H
J Biochem. 1981 Jul;90(1):163-75. doi: 10.1093/oxfordjournals.jbchem.a133446.
Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagent bind, induced by Ca2+ binding to the high affinity Ca2+-binding sites is very different from that induced by Mg2+ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca2+ binding to the low affinity Ca2+-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca2+ binding to the low affinity Ca2+-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca2+ binding to or removal from the high affinity Ca2+-binding sites by following the fluorescence intensity change of ANM-labeled TN-C, respectively. THe kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinty Ca2+-binding sites (rate constants, 3.7-157 s-1) are slower than the kinetic processes associated with the low affinity Ca2+-binding sites (rate constants, equal to or higher than 230 s-1) in the absence of MgCl2. In the presence of 2 mM MgCl2, a new slow phase (rate constant, 10-16 s-1) appears in the kinetic processes associated with the low affinity Ca2+-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl2. Kinetic properties associated with the high affinity Ca2+-binding sites are not essentially altered by addition of 2 mM MgCl2 to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s-1).
用N-（对-（2-苯并咪唑基）苯基）-马来酰亚胺（BIPM）标记的骨骼肌肌钙蛋白C（TN-C），在Ca²⁺结合到TN-C的高亲和力Ca²⁺结合位点（位点III和IV）时荧光增加约5%，在Mg²⁺结合到这些位点时荧光增加82%。用N-（1-苯胺基萘基-4）马来酰亚胺（ANM）标记的TN-C，在Ca²⁺结合到高亲和力Ca²⁺结合位点时荧光降低约26%，在Mg²⁺结合到这些位点时荧光增加22%。这些发现表明，Ca²⁺结合到高亲和力Ca²⁺结合位点所诱导的、马来酰亚胺试剂结合位点Cys-98周围的环境变化，与Mg²⁺结合到相同位点所诱导的环境变化非常不同。这些染料-蛋白质缀合物在Ca²⁺结合到低亲和力Ca²⁺结合位点（位点I和II）时不显示荧光强度变化。丹磺酰氮丙啶（DANZ）标记的TN-C在Ca²⁺结合到TN-C的低亲和力Ca²⁺结合位点时荧光增加超过100%。因此，通过分别跟踪ANM标记的TN-C的荧光强度变化，我们可以观察到TN-C在Ca²⁺结合到高亲和力Ca²⁺结合位点或从该位点去除时所经历的动力学过程。在不存在MgCl₂的情况下，与高亲和力Ca²⁺结合位点的Ca²⁺结合和去除反应相关的荧光强度变化的动力学过程（速率常数，3.7 - 157 s⁻¹）比与低亲和力Ca²⁺结合位点相关的动力学过程（速率常数，等于或高于230 s⁻¹）慢。在存在2 mM MgCl₂的情况下，除了在不存在MgCl₂时已经观察到的快速相之外，在与低亲和力Ca²⁺结合位点相关的动力学过程中出现了一个新的慢相（速率常数，10 - 16 s⁻¹）。向系统中添加2 mM MgCl₂基本上不会改变与高亲和力Ca²⁺结合位点相关的动力学性质，但ANM标记的TN-C显示出较低的速率常数（0.67 - 26 s⁻¹）。
