Conformational effects in the high-performance liquid chromatography of proteins. Further studies of the reversed-phase chromatographic behavior of ribonuclease A.

This paper is a continuation of an examination of the conformational effects of ribonuclease A (RNase A) in reversed-phase liquid chromatography. RNase A is a particularly good example of reversible conformational refolding during chromatographic elution. Absorbance ratio measurements with a photodiode-array detector are consistent with a two-state model in which an early eluted broad band is associated with the folded or native state and a late eluted sharp band is associated with an unfolded state. By varying the mobile phase flow-rate while maintaining the mean capacity factor (k') of the protein in gradient elution constant, the apparent rate constant of refolding in the mobile phase was measured and found to be comparable with that reported in the literature. It has been further shown that, as the gradient steepness parameter b or mean k' value is altered, the apparent rate constant also changes, as a result of variation in mobile phase composition. The apparent rate constant as a function of temperature was also found to reach a maximum at 20 degrees C. Based on the results of this model system, it is possible to predict optimum conditions for elution of species with sharp elution peaks when reversible refolding takes place in the column.