An investigation of the use of nile red as a long-wavelength fluorescent probe for the study of alpha 1-acid glycoprotein-drug interactions

J Pharm Biomed Anal. 1995 Jul;13(8):1011-7. doi: 10.1016/0731-7085(95)01524-o.

Abstract

Spectrofluorimetry in the long-wavelength region of the electromagnetic spectrum (600-1000 nm) is a fairly recent development in photoluminescence spectroscopy, which has numerous advantages over measurements in the more conventional ultraviolet and visible spectral region. 9-Diethylamino-5H-benzophenoxazine-5-one (Nile Red) is an unchanged, hydrophobic molecule, and long-wavelength fluorescence of which is strongly influenced by the polarity of its environment. When Nile Red was added to solutions of alpha 1-acid glycoprotein (Orosomucoid. OMD), it showed an enhancement in fluorescence intensity and a shift to blue in emission wavelength, suggesting it was binding hydrophobically to a non-polar site on the protein. The association constant (12,261,000 +/- 900,000 M-1) and number of binding sites (0.746 +/- 0.044) were calculated for the probe. Upon addition of both acidic and basic drugs, the Nile Red fluorescence reverted to its unbound form, indicating that OMD probably has one high-affinity, wide and flexible binding area for such drugs. Possible enantiomeric selectivity was shown with ephedrine, and the association constant determined for a racemic mixture of propranolol was found to be comparable to other values obtained with alternative, more conventional techniques.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Buffers
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Orosomucoid / chemistry*
  • Oxazines / chemistry*
  • Propranolol / analysis
  • Scattering, Radiation
  • Spectrometry, Fluorescence
  • Stereoisomerism

Substances

  • Buffers
  • Fluorescent Dyes
  • Orosomucoid
  • Oxazines
  • Propranolol
  • nile red