Analysis of free solute fractions and solute-protein interactions using ultrafast affinity extraction and affinity microcolumns

This dissertation describes the use of a high-performance affinity chromatography method based on ultrafast affinity extraction and microcolumns to study biological interactions. In the first project, a new method was created and based on ultrafast affinity extraction to determine both the dissociation rate constants ( kd) and association equilibrium constants (K a) for drug-protein interactions in solution. Various conditions to optimize the use of ultrafast affinity extraction for equilibrium and kinetic studies were considered. The objective of the next portion of this dissertation was to develop a chromatographic approach to measure free drug fractions in more complex samples. This was accomplished by combining ultrafast affinity extraction with a multi-dimensional HPAC system. In the first project in this section, the target of interest was R- and S-warfarin, which have slightly different binding strengths for the serum transport protein human serum albumin (HSA). A multi-dimensional HPAC system was developed to study the binding of each enantiomer with HSA. This system was used to simultaneously measure the free fraction of each enantiomer and its Ka value with HSA. The second project used ultrafast affinity extraction and a multi-dimensional affinity system to measure the free fractions and global affinity constants of several sulfonylurea drugs in the presence of normal HSA or glycated HSA. A third project used a similar approach to measure the free fractions and Ka value of various drugs in serum. The next project used ultrafast affinity extraction to study the interactions of a steroid hormone (i.e., testosterone) with its serum transport proteins, HSA and sex hormone binding globulin (SHBG). Both the kd and Ka values for these systems were determined. The free fractions of testosterone in samples containing HSA or SHBG at physiological concentration were also estimated. The last project sought to develop a method to increase the binding capacity and activity of proteins in small affinity columns. This was accomplished by combining a traditional covalent immobilization method with protein cross-linking/modification. It was found that up to a 75–113% increase in total protein content could be obtained by this method when compared with more traditionally prepared supports