Resonance Raman spectroscopic study on chiral aggregation of bilirubin-bovine serum albumin complex formed at liquid/liquid interface.

Resonance Raman spectroscopy assisted by centrifugal liquid membrane/circular dichroism (CLM-CD) and UV/Vis absorption spectroscopies was applied to measure the binding state of bilirubin (BR) in the complex with bovine serum albumin (BSA) formed at a heptane/water interface. The bisignate Cotton effects in the interfacial CD spectra and the red shift and linewidth increase of the BR absorption band around 450 nm indicated the formation of the BR-BSA complex at the interface and the chiral conversion of BR molecules in the aggregates. The resonance Raman spectra of BR observed at the interface suggested that the interfacial BR-BSA complex formed during the initial 15 min after the contact of the two phases had a similar structure with that in solution, but after 15 min were forming aggregates coexisting with solid micro-particles. These experimental results strongly suggested that the chiral interconversion of BR from (P+) conformation to (M-) conformation in the interfacial complex was accompanied by aggregation of the BR-BSA complexes. In the present study, resonance Raman microscopic spectrometry was proved to be highly useful for characterizing the solid like aggregate formed at the liquid/liquid interface.