Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy

Abstract

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.