Fluorescence Titration and Fluorescence Stopped-Flow Studies on Skeletal Troponin C Labeled with Fluorescent Maleimide Reagent or Dansylaziridine

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Abstract
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 reagents 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 and those upon Ca2+ binding to or removal from the low affinity Ca2+-binding sites by following the fluorescence intensity change of DANZ-labeled TN-C, respectively. The kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinity 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 2mM 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).