Search published articles
Showing 2 results for Aequorin
, , Reza Hassan Sajedi, , , ,
Volume 5, Issue 1 (11-2014)
Abstract
Mnemiopsin, a Ca2+-regulated photoprotein from ctenophore Mnemiopsis leidyi, as coelentrate photoproteins emits flash blue light upon reacting with coelenterazine. In contrast to coelenterate photoproteins, there is a little information about the structure of chromophore binding site and bioluminescence mechanism in ctenophore photoproteins. In this study, three important amino acid residues in coelenterazine binding cavity of mnemiopsin were substituted by corresponding residues in the well-known coelentrate photoproteins. W59K, N105W and L127W mutants were constructed and characterized for investigation of hydrogen bond network around the important rings of coelenterazine. All three mutants are completely inactivated. In addition, the results of structural studies including CD, intrinsic and extrinsic fluorescence together with theoretical studies showed that these mutants, especially for N105W and L127W, have found different structural features. These results suggest the presence of the residues in binding cavity and/or a mechanistic role for these residues. It seems that arrangement of amino acid residues in the binding cavity of coelenterate and ctenophore photoproteins are different, so that the replacement of these residues with their corresponding residues in other group (such as mutations in this study) perturbs the structural integrity needed for bioluminescence activity.
H. Rahmani, R. H.sajedi ,
Volume 10, Issue 4 (12-2019)
Abstract
Aims: Aequorin as a bioluminescence protein due to ease of use, non-toxic, and high capability of detecting has long been the interest of researchers. The aim of this study was to design a method for accurate and simple detection of important therapeutic agents using a bioluminescence inhibition based assay by using aequorin.
Materials & Methods: In this study, important drugs in therapeutic monitoring with structural similarity to Coelenterazine, were selected and their interaction with aequorin was investigated. Further, the conditions of the bioluminescence assay were optimized to achieve the lowest detection limit.
Findings: Among the drugs whose effects have been tested on aequorin, the only benserazide resulted in inhibition of the bioluminescence activity. This analyte can significantly reduce the bioluminescence of aequorin in a concentration-dependent manner. The best dose-response curve was obtained and IC50 of 0.26µM was calculated. The linear calibration curve was obtained in a range of about 100 to 1500nM with LOD and LOQ of 79 and 260nM, respectively. Furthermore, we demonstrated the application of the approach in human serum samples with a recovery of 97%. Guddem-Schild graph was plotted to determine the mechanism of inhibition which indicated that the IC50 of benserazide changed in the presence of different concentrations of Coelenterazine.
Conclusion: The proposed method can be used for measuring benserazide which can easily be applicable for real samples. Also, the results show that benserazide inhibits the bioluminescence activity of aequorin by competitive inhibition.
