Conformational effects in the reversed-phase liquid chromatography of ribonuclease A.
This paper examines the reversed-phase liquid chromatographic behavior of ribonuclease A (RNase) using an n-butyl chemically bonded phase and a gradient of 10 mM H3PO4 and l-propanol. At a column temperature of 25 degrees C, a broad band followed by an overlapped late-eluting sharp peak is observed. As the temperature is raised, the sharp peak grows at the expense of the broad band until at 37 degrees C, only a single narrow-eluting band is found. Using an absorbance ratio of A288/A254, it is demonstrated that the broad band represents a folded or native state of RNase and the late-eluting band a denatured state. Based on postcolumn absorbance ratio changes in the denatured state as a function of time and the known behavior of the protein, reversible refolding or renaturation is proposed to take place in solution. RNase is denatured upon adsorbing to the bonded phase, and upon migration down the column, reversible refolding takes place in the mobile phase. The relaxation time for native state formation is assumed to be comparable to the time spent by RNase in the mobile phase. As temperature is raised, both the native and denatured states exist at equilibrium in solution, thus slowing the refolding process, until at 37 degrees C only the denatured peak appears. Changes in peak shape with flow rate provide further evidence for this model. The use of HCl or H2SO4 instead of H3PO4 yields similar results except that the temperature at which only the denatured peak is observed follows the order of salt stabilization of the native state.
D000433:1-Propanol; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004586:Electrophoresis / Q000379:methods; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics
D000433:1-Propanol; D012259:Ribonuclease, Pancreatic