title: Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.
abstract: We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.
authors.affiliation: a Chemistry Department , University of Calcutta , Kolkata , 700009 , India .
authors.forename: Malay
authors.initials: M
authors.lastname: Patra
authors.forename: Madhurima
authors.initials: M
authors.lastname: Mitra
authors.forename: Abhijit
authors.initials: A
authors.lastname: Chakrabarti
authors.forename: Chaitali
authors.initials: C
authors.lastname: Mukhopadhyay
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D004912:Erythrocytes / Q000737:chemistry*; D005456:Fluorescent Dyes; D006041:Goats; D057927:Hydrophobic and Hydrophilic Interactions; D008840:Microfilament Proteins / Q000737:chemistry*; D062105:Molecular Docking Simulation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011721:Pyrenes / Q000737:chemistry; D012756:Sheep; D013049:Spectrin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D015081:2-Naphthylamine; D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D011721:Pyrenes; D013049:Spectrin; C027132:1-anilino-8-naphthalenesulfonate; C038517:prodan; C030984:pyrene