Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces

Area of Science:

• Biophysical Chemistry
• Macromolecular Science
• Spectroscopy

Background:

• Indicator dyes exhibit altered pK values when bound to charged macromolecules.
• The difference between bound (pKb') and free (pKf') dye pK values, termed increment incrementpK, is investigated.

Purpose of the Study:

• To determine if increment incrementpK changes accurately reflect electrostatic potential changes at binding sites.
• To utilize spectrophotometric and binding experiments to study dye-macromolecule interactions.

Main Methods:

• Spectrophotometric analysis of Chlorophenol Red and Phenol Red interactions with human serum albumin.
• Binding experiments with cationic micelles (cetyltrimethylammonium chloride, cetylpyridinium chloride).
• Investigation of pH and ionic strength effects on dye binding and pK values.

Main Results:

• Increment incrementpK decreased with reduced pH in serum albumin solutions, correlating with increased protein positive charges.
• Binding affinity and increment incrementpK showed opposite pH-dependent trends, with basic dyes being more sensitive.
• Micelle binding demonstrated preferential uptake of basic dye forms, and increased ionic strength yielded positive increment incrementpK values.

Conclusions:

• Observed increment incrementpK values reliably indicate changes in the electrostatic potential at binding sites.
• The intrinsic pK of the dye is minimally affected by these electrostatic interactions.
• The method shows potential for measuring electrostatic potential changes at cell membrane binding sites.