AL Pressure-induced protein denaturation in liquid


chromatography An LC sample is generally prepared and stored at atmospheric pressure until it is switched into the flow stream and transported to the column. Upon injection, the pressure on the sample increases rapidly to the system pressure, which can range from 10 to 2000 bar. Small molecules and some peptides and smaller proteins are robust and are not affected by pressure. However, robustness decreases with increasing protein size; for example, insulin (small) is more robust than hemoglobin, which in turn is more robust than multimeric proteins such as IgMs (very large, with several subunits).


Pressure eff ectively denatures proteins since interruption of the native structure usually leads to a reduction in system volume. The presence of internal cavities within the folded protein structure particularly favors pressure-induced unfolding.


The column packing and mobile phase can interact with the protein and may alter the mechanism of unfolding by lowering the activation energy of the unfolding reaction. Fekete and Guillarme studied RPLC, with reten- tion based on hydrophobic interactions. Some proteins have hydrophobic patches on the surface. These are often necessary for their location and function. By way of contrast, other chromatographic modes, such as ion exchange, prefer interactions (attractive or repulsive) with ionophores. Steric exclusion chromatography (SEC) should have the lowest interaction with the stationary phase. Studies in SEC mode would be an interesting extension of Fekete and Guillarme’s work.


Chromatographic procedure The chromatographic process can be explained in three parts.


Top of the column, including the inlet frit Upon injection, the sample encounters the frit followed by the top of the chromatographic bed. A protein analyte endures the full system pressure. With sub-2-μm column packings, the pressure can be in the low-kbar range.


Rates of unfolding are rarely measured, however. A paper by Broom et al.6 (University of Waterloo, Ontario, Canada) reports that protein-unfolding rates correlate strongly with folding rates, which restores the native struc- ture. In a related report by Yanxin Liu et al.7


(University of Illinois, Urbana,


Ill.), pressure-jump was used to study the denaturation and refolding of a lambda receptor. The formation of helices proceeded stepwise and was complete in 19 μsec. A second folding phase of about 1.5 msec resolved errors in the μsec helix formation.


Thus, for some proteins, there is a possibility that pressure-induced denaturation quickly leads to a denatured state, which may expose hy- drophobic surfaces to interaction with the hydrophobic stationary phase. Fekete and Guillarme studied this eff ect by increasing the fl ow rate from about 100 μL/min (atmospheric P) to 1400 μL/min (750 bar). They discov- ered that retention increased 240% for lysozyme and 480% for interferon alpha-2A. Since the stationary phase is hydrophobic, the retention may be due to increased exposure of hydrophobic patches of the analyte. Small molecules, on the other hand, show only a small (25–100%) increase when


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