May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
ANS Fluorescence in Ion Pairing- Model for External Binding Sites of Proteins
Author Affiliations & Notes
  • B. J. Glasgow
    JSEI/UCLA, Los Angeles, California
    Pathology & Ophthalmology,
  • O. K. Gasymov
    JSEI/UCLA, Los Angeles, California
  • Footnotes
    Commercial Relationships B.J. Glasgow, None; O.K. Gasymov, None.
  • Footnotes
    Support NIH Grant EY11224 and the Edith and Lew Wasserman Professorship
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1900. doi:
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      B. J. Glasgow, O. K. Gasymov; ANS Fluorescence in Ion Pairing- Model for External Binding Sites of Proteins. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1900. doi:

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: ANS (8-anilino-1-naphthalenesulfonic acid) is widely utilized to probe hydrophobic binding sites with functional correlation in many proteins including lens crystallins and tear lipocalin. ANS is believed to fluoresce only when bound to hydrophobic sites. Recent work indicates ANS binds cationic groups of proteins through ion pairing. Little data exists on the fluorescent properties of ANS in these conditions. ANS interactions with Arg and Lys derivatives were studied for changes in fluorescence and secondary structure, which typify the external binding sites of proteins.

Methods:: Fluorescence spectra were obtained of ANS and its analog 1NPN titrated with synthetic derivatized amino acids at pH 7.3 and pH 3.0 to determine the effect of charge on the interaction with the sulfonate group of ANS. Fluorescence decays of ANS and 1NPN was measured in solutions of derivatized amino acids using a fluorescent lifetime instrument. Fluorescence intensity and lifetime parameters were analyzed with model free maximum entropy method and a multiexponential decay law. Circular dichoism spectra were recorded, using poly-Arg with varying concentrations of ANS.

Results:: Absence of the sulfonate group (1NPN) results in a 10 fold prolonged fluorescent lifetime compared to ANS. Increased fluorescence intensity and a blue shift of the emission maxima were observed for ANS interacting with derivatives of Arg with a negative charge. An ion pairing between Arg (or Lys) and the sulfonate group of ANS reduce the intermolecular charge transfer rate constant that leads to enhancement of fluorescence. A positive charge near the NH- group of ANS, however, changes the intramolecular charge transfer process producing a blue shift of fluorescence. The Arg side chain compared to that of Lys more effectively interacts with both the NH- and sulfonate groups of ANS. The dissociation constants for ANS binding to poly-Arg and poly-Lys were estimated to be about 1.7 mM and 2.6 mM, respectively. ANS binding to poly-Arg induced circular dichroic minima at 207 and 223 nm indicative of a random coil-alpha helix transition in poly-Arg.

Conclusions:: Ion pairing of ANS with charged residues increases fluorescence intensity and lifetime. This model suggests that induced secondary structure may result from ANS binding in proteins with sequentially positioned Arg and Lys. Utilization of ANS binding to proteins solely as a hydrophobicity probe may overlook ion pairing of ANS to external sites. The use of steady state and lifetime fluorescence parameters of ANS is a potential tool to characterize the external binding sites of proteins.

Keywords: protein structure/function • lipids • cornea: basic science 

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