Surface‐enhanced resonance Raman study of avidin–dye interactions: A model for chromophore‐containing proteins

Abstract

AbstractResonance Raman (RR) and surface‐enhanced resonance Raman (SERR) spectroscopy were used to study the behavior of 2‐(4′‐hydroxyphenylazo)benzoic acid (HABA) at different pH values (2.7–11.8) in solution and bound to avidin. Major changes in the RR spectrum of the dye are observed near pH 8.0 and 3.0. These pH values are close to the pKa values for the phenolic and carboxylic acid functional groups of HABA and reflect deprotonation of these groups. In addition, however, tautomeric forms of the dye are possible within each pH range and these can be identified from the characteristic vibrational modes of the azo and hydrazone forms of HABA. Similar changes in the SERR spectrum of HABA were also observed as a function of pH. However, a comparison of the RR and SERR spectra has shown that the tautomeric equilbrium is shifted toward the hydrazone form on the Ag surface compared with the effect in solution.The RR and SERR spectra of the HABA‐avidin complex are nearly identical and indicative of the hydrazone form of the dye. Hence the complex is not dissociated on the Ag surface. This finding is encouraging with regard to the application of SERR techniques to the study of native proteins containing chromophores.