A chemometric approach to the estimation of the absorption spectra of dye probe merocyanine 540 in aqueous and phospholipid environments.

Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.