Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography. On-line spectroscopic and chromatographic characterization.
The thermal behavior of a series of standard proteins in hydrophobic interaction chromatography on a previously developed weakly hydrophobic ether-bonded phase column has been studied. Depending on the temperature and protein, conformational changes can occur in the chromatographic system. Methods for recognizing these conformational effects are presented, including retention and peak width variations with temperature, and Z values (the slope of the plot of log k' vs. log %B solvent. The Z value is shown to be a general index characterizing protein retention as a function of salt concentration. In addition, on-line UV spectroscopic analysis, (absorbance ratios and second derivative spectroscopy) with a photodiode array detector, is shown to corroborate chromatographic trends. Lysozyme maintains a stable conformation over the temperature range 10-40 degrees C, whereas beta-lactoglobulin A has a conformational transition between 25 degrees C and 40 degrees C. Calcium-depleted alpha-lactalbumin, a rather labile species, maintains a stable conformation up to ca. 20 degrees C, and then undergoes structural changes. Finally, cytochrome c appears to be relatively stable up to ca. 65 degrees C. Since conformational changes for this protein occur at ca. 35 degrees C on more hydrophobic phases, the extent of hydrophobicity of the stationary phase is important for maintenance of the native state. Based on this work, hydrophobic-interaction chromatography at sub-ambient temperatures appears promising.
D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000032:analysis; D007782:Lactoglobulins / Q000032:analysis; D009113:Muramidase / Q000032:analysis; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D003574:Cytochrome c Group; D007782:Lactoglobulins; D011506:Proteins; D009113:Muramidase