Date of Thesis

Summer 2014


Bile salts are biomolecules that are produced in the liver and are responsible for a range of functions in the process of digestion, primarily the emulsification of dietary fat and fat-soluble vitamins. Despite their importance in biological chemistry, the structure and dynamics of bile salt aggregation are not well understood. The efforts described herein attempt to enhance the understanding of cholate aggregation numbers (AN), critical micelle concentration (CMC), micellar structure(s), and interactions with a binaphthyl probe molecule. Cholate is the most common bile salt in mammals and is, therefore, a decent model for describing bile salt aggregation. CMC determination is achieved by observing the 1H NMR chemical shift perturbation of 1,1’-binaphthyl-2,2’-diyl hydrogen phosphate (R,S-BNDHP), a probe molecule for bile salt aggregation, when exposed to increased concentrations of sodium cholate. Using NMR and a phase-transition model to determine CMCs for pH 12.0 sodium cholate results in the observation of three unique CMC values at 6.1, 11.0, and ~25 mM. Using 1H-13C heteronuclear single quantum coherence (HSQC) spectroscopy, a two-dimensional NMR experiment, it appears that anti-parallel cholate dimers are not strictly collinear, but rather a skew exists between the two-cholate monomers. The existence of a skew is surprising as it would be incongruent with a well-known model of bile salt aggregation proposed by Donald Small proposed in 1968. HSQC also showed evidence that R- and S-BNDHP attack different edges of a cholate aggregate, possibly explaining the chiral selectivity exhibited by sodium cholate aggregates in earlier micellar electrokinetic chromatography experiments and confirming previous two-dimensional nuclear Overhauser effect (NOE) NMR data. HSQC data also suggest evidence for the interactions responsible for the aggregation of predicted aggregates by Small’s model. High-resolution negative ion electrospray ionization mass spectrometry (ESI-MS) data suggest that cholate is capable of forming several aggregates of sufficient stability for mass analysis, the most massive of which is an aggregate with an aggregation number of 18. With these data it is clear that this system has several complexities that affect aggregation that may not be accounted for in previous bile salt aggregation models.


Bile Salt, Aggregation, NMR, Cholate, Nuclear Magnetic Resonance, Mass Spectrometry

Access Type

Masters Thesis

Degree Type

Master of Science



First Advisor

Timothy G. Strein

Second Advisor

David Rovnyak