[Fluorescence of Tb(3+) -calmodulin complex and its analytical application].
Calmodulin (CaM) is a ubiquitous Ca(2+) -binding protein of eukaryotes, and regulates a broad spectrum of fundamental cellular processes. CaM harbors four binding domains, among which domains I, II and III contain no tyrosine, only domain IV has one tyrosine for plant species. In contrast to mammals, plants express numerous CaM isoforms that exhibit differential activation or inhibition of CaM dependent enzymes in vitro. In the present study, the isoform II of Arabidopsis thaliana CaM was used to test the binding properties of metal ion to CaM by Tb3+ fluorescence. The increase in fluorescence was monitored at 545 nm as a function of the number of Tb3+ bound to CaM using direct (221 nm) and indirect (280 nm ) excitation. Upon direct excitation of Tb(3+) -CaM, the fluorescence of Tb3+ increased markedly, and one of the pathways of energy dissipation of the excited state of Tb3+ was energy transfer to the vibrational levels of water molecules in the hydration sphere around the Tb3+ ion. When waters of hydration were removed as a result of Tb3+ binding to CaM, an increase in rate constants of luminescence was observed. The titration curve with direct excitation increased up to 4 mol of Tb3+/mol of CaM before the onset of a plateau, in agreement with the expected maximum of four binding sites. Using indirect excitation at 280 nm, the resultant titration curve was sigmoid, albeit with less fluorescence intensity, also reached a maximum at a ratio of 4 mol of Tb3+/mol of CaM, in which the first phase exhibits an end at 2 : 1, and in this phase there was only a small increase in Tb3+ fluorescence. The fact that only the second pair of added Tb3+ shows a large enhancement in Tb3+ fluorescence suggests that it is Tb3+ bound to the low affinity sites that can accept energy from tyrosine group. The turning points of fluorescence titration curves were used to estimate the concentrations of CaM on the base of CTb3+ = 4c(CaM).
Xiao
Feng-Juan
FJ
Material Branch of College, College of Railway, Shijiazhuang 050043, China.
Liu
De-Long
DL
Bai
Juan
J
Sun
Da-Ye
DY
D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry*; D056890:Eukaryota; D005453:Fluorescence; D011487:Protein Conformation; D013725:Terbium / Q000737:chemistry* / Q000378:metabolism
D002147:Calmodulin; D013725:Terbium