| The overall goal of this work was to develop a better understanding of how and why proteins unfold on RPC media, and by extension on hydrophobic surfaces generally, by exploring how protein structural change on RPC media relates to protein physical properties. The ultimate goal is to be able to determine, a priori, how a particular protein will behave structurally by examining its physical properties. A variety of topics was explored in striving towards this goal including: the development of an updated algorithm for protein secondary structure estimation from amide I band Raman spectra, the development of a physical and thermodynamic property database for a set of 34 well-characterized proteins, a batch adsorption study of 9 proteins on C18 reverse phase chromatographic media including adsorbed amount and the secondary structure consequences of exposure to media and corresponding mobile phases, and a correlation study of adsorbed amount and structure perturbations with the protein properly database.; Data analysis methods for protein Raman spectroscopy, a technique that allows us to obtain secondary structure information from proteins in a wide variety of sample states, including proteins adsorbed onto RPC media, were improved. We developed new secondary structure reference spectra for the Holistic Estimation Approach to Secondary Structure (HEAPSS) (Sane et al. 1999a), based on Dictionary of Secondary Structure of Proteins (DSSP) (Frishman and Argos 1995) estimates of secondary structure from x-ray crystallography data. We were able to develop three equally accurate sets of secondary structure reference spectra for HEAPSS with varying levels of parsing of tune and unordered secondary structure, ranging from combined turn and unordered structure to divided turn and unordered structure with differing reference spectra for varying type of turns, allowing the choice of the most appropriate level of detail for structure parsing for protein Raman spectra.; A varied set of 34 well-characterized proteins was selected as a basis set for studying protein structural change on RPC and a database of protein properties of these proteins was developed, including properties calculated directly from a protein's Protein Data Bank (PDB) entry (Berman et al. 2000) and thermodynamic properties. In order to include measured thermodynamic properties, namely melting temperatures, of proteins in conditions relevant to RPC in that database, we developed a method for collecting and analyzing far-UV CD melting curves for protein stability information. Correlations between properties in the database in were explored in order to establish the linear independence of the database properties.; A batch adsorption study of a small subset of the study set proteins was performed on RP C18 media. Mobile phase carrier (25 mM potassium phosphate at pH 7) and modifier (10 to 50 vol% isopropanol) were selected in an attempt to make the data as pharmaceutically relevant as possible. Absorbance measurements at 280 nm were collected of the loading supernatant and desorbed protein solution to determine adsorbed and desorbed concentrations. CD and Raman spectra for secondary structure estimation were collected from relevant samples during each batch experiment. Significant correlations were found between amount of protein adsorbed and the structural changes due to initial contact with the media, being adsorbed, and remaining after desorption. Furthermore, we identified a common mode of structural perturbation wherein changes in helix content and changes in sheet content are strongly negatively correlated: gains in sheet content occur at the expense of helix content and vice versa.; Relationships between batch study results and protein properties were explored based on single property and aggregated property information to move towards determining which sets of protein property information would be predictive of protein behavior in hydrophobic chromatography. We have not... |
