Spectral Studies On The Interaction Of Truncated Euplotes Octocarinatus Centrins With Metal Ions

Centrins are an acidic protein of 20 kDa, which belongs to the highly conserved EF-hand CaM super family of Ca²⁺-binding proteins. It is a ubiquitous highly conserved protein in diverse evolutionary lineages, including algal, higher plants, invertebrate and mammalian cells. Centrins play a fundamental role in centriole duplication and contraction of center basal body fibrous. Up to now, the studies on the centrins have still been concentrated on the structure and function.In recent years, with the exploitation of rare earth resources, rare earth ions are becoming closely related with people's daily life. So, it is imperative for us to understand the safety and biological effects on organisms. Calcium has the similarity in coordination chemistry with rare earth elements. Thus, the biological effects of calcium must be influenced owing to the more and more rare earth elements coming into food cycle. Centrins are a subfamily within the superfamily of Ca²⁺-modulated proteins. Structurally, it contains four helix-loop-helix motifs or EF-hands, each of which has the potential to bind one calcium atom. So we infer that centrins will be the carrier by which rare earth ions achieve their biological effect in organisms.Euplotes octocarinatus, a unicellular protozoa, locate in a special phylogenic degree showing a number of exceptional features. It can help to clarify the natural of the progress of life. In this study, firstly, we have expressed and purified the fragments of the Euplotes octocarinatus centrin P₁₂(N-EoCen) and P₂₃ by biological engineering. Some parameters of P₁₂ and P₂₃ were predicted by GenePro, such as molecular weight, isoelectric point, net charge, the extraction coefficient, et.al., which aim to provide some theoretical guidance for the succeeding experiential research.Secondly, the binding properties of P₁₂ for Tb³⁺, Ca²⁺ and Mg²⁺ were studied by terbium fluorescence probe and ionic competition, and the condition binding constant of Tb₂-P₁₂, the relative binding constants ofCa2-Pi2, Mg2-Pi2 were obtained. To deeply investigate the interaction of Tb3+ and Pi2, the thermodynamic parameters were gotten by the Van't Hoff equation. The result shows thatZlrG< 0, demonstrated that the reaction is spontaneous, while z)rH < 0, ZlrS > 0 indicated that the binding force act of Tb3+ and P!2 is major electrostatic interaction.Lastly, TNS is one of a class of compounds which do not fluoresce in water but fluoresce strongly both in organic solvents and when bounds to certain native proteins. The conformational change of the P]2> P23 induced by the different metal ions were studied using TNS as a probe. The results demonstrate that the extent of the conformational change by binding the metal ions is proportional to the ion potential. The conditional binding constants of TNS binding M2-P12 or M2-P23 were calculated by the same method described above. Resonance light-scattering (RLS) experiments show that P12 plays an important role in protein polymerization.