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Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
In vitro relationship between serum protein binding to beta-carboline alkaloids: a comparative cytotoxic,
Tapas Ghosh1, Sarita Sarkar1,2, Paromita Bhattacharjee1
1Department of Zoology, University of Kalyani, Nadia, West Bengal, India.
Abstract:
The work highlighted interaction of harmalol, harmaline and harmine with human serum albumin by biophysical and biochemical assays. Presence of serum protein in the media negatively affects the cytotoxicity of the alkaloids. MTT assay indicates concentration-dependent growth inhibitory effect of the alkaloids on A375, MDA-MB-231, HeLa, A549, ACHN and HepG2 cell, having maximum cytotoxicity with GI50 value of 6.5 μM on ACHN by harmine in 1% of fetal bovine serum. Detail cytotoxic studies on ACHN cell by harmine, the most cytotoxic among the three, reveal nucleosomal fragmentation, formation of comet tail, generation of reactive oxygen species, decreased mitochondrial membrane potential, up regulation of p53, caspase 3 and significant increase in G/M population that made the cancer cells prone to apoptosis. Furthermore, the findings unequivocally pointed out that harmine binds strongly to the protein with a binding constant of 5.53 × 104 M-1 followed by harmaline and least with harmalol. Thermodynamic results revealed enthalpy dominated, entropy favored, 1:1 binding. Molecular docking and circular dichroism suggested changed conformation of protein by partial unfolding on complexation. Further supported by infrared analysis where protein secondary structure was altered with a major decrease of α-helix from 53.68% (free protein) to 8-11% and change in β-sheet from 25.31% (free protein) to 1-6% upon binding, inducing partial protein destabilization. Site markers demonstrated site I (subdomain IIA) binding of the alkaloids to the protein. The results serve as data for the future development of serum protein-based targeted drugs. AbbreviationsCD: circular dichroism; FBS: fetal bovine serumFRETForster resonance energy transferFTIRFourier transform infraredHSAhuman serum albumin; ROS: reactive oxygen speciesCommunicated by Ramaswamy H. Sarma.
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