The effect of pH and temperature on the conformational stability of recombinant human interleukin-2

Date of Completion

January 1999


Chemistry, Pharmaceutical|Health Sciences, Pharmacy




The goal in designing a suitable drug product is to provide optimum conditions that result in the maximum stability of the drug. The structure of protein drugs can be greatly affected by the solution environment such as pH and temperature, which can decrease their shelf-life, rendering them therapeutically inactive. The objective of this research was to investigate the pH and temperature induced conformational changes in recombinant human interleukin-2 (rhIL-2). ^ The effect of pH on the structure of rhIL-2 was investigated by studying the unfolding of the protein and the resulting changes in the secondary structure. The 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence showed that, rhIL-2 unfolds as the pH is decreased from 6.5 to 2.7. The tryptophan environment remains relatively intact during the unfolding. The molecule partially refolds to its native structure when the pH is adjusted back to 6.5. Storage of rhIL-2 at pH 2.7 affects its secondary structure and leads to the aggregation of the protein. The rate of aggregation was lowest around pH 4.5. ^ The effect of temperature on the conformation of rhIL-2 was investigated. As the temperature reaches 44°C, rhIL-2 partially unfolds and results in an increase in the surface hydrophobicity of the protein as shown by ANS fluorescence. This was confirmed by Hydrophobic Interaction Chromatography data. During partial unfolding the protein retains most of its helical structure. However, due to the exposure of the hydrophobic regions to the solvent, rhIL-2 aggregates under these conditions. ^ The presence of ANS molecules during thermal cycle was found to induce aggregation in rhIL-2. This lead to precipitation of the ANS-rhIL-2 complex. The results demonstrate the limitation of using this probe in studying protein conformational changes during thermal cycles. ^ In conclusion, changes in the pH and temperature both lead to unfolding of rhIL-2, without affecting the tryptophan environment. Our results showed that studying surface hydrophobicity of proteins is a useful approach, which enables us to detect conformational changes not shown by tryptophan fluorescence or secondary structure. Meanwhile, extrinsic fluorescence probes should be used with caution since these agents can induce the aggregation of unfolded proteins. ^