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Temperature- and pH-induced multiple partially unfolded states of recombinant human interferon-alpha2a: possible implications in protein stability


, : Temperature- and pH-induced multiple partially unfolded states of recombinant human interferon-alpha2a: possible implications in protein stability. Pharmaceutical Research 20(11): 1721-1729

Purpose: To study the effect of solution conditions on the structural conformation of recombinant human interferon-alpha2a (IFNalpha2a) to investigate its tendency to form partially unfolded intermediates. Methods: The structural properties of IFNalpha2a were studied at various pH values (2.0-7.4) and temperatures (5degreeC-80degreeC) using Trp fluorescence emission, fluorescence quenching, near- and far-UV circular dichroism (CD) spectroscopy, and DSC. Results: Fluorescence intensity measurements as a function of temperature indicated the onset of the thermal unfolding of IFNalpha2a, denoted by Td, around 60degreeC above pH 4.0. Td was not observed at pH 3.5 and below. Acrylamide and iodide quenching studies indicated partial unfolding of protein with decrease in pH and with increase in temperature up to 50degreeC. Near-UV CD studies indicated a significant loss in the tertiary structure of protein on increase in temperature from 15degreeC to 50degreeC at all solution pHs. DSC scans supported results obtained from fluorescence and CD studies at pH 4.0 and below. DSC, however, was insensitive to changes that occurred at moderate temperatures at pH 5.0 and 7.4. Conclusions: IFNalpha2a has a tendency to acquire multiple partially unfolded states with structural conformations sensitive to solution pH and temperature. These states were formed at moderate temperatures, and it is speculated that these partially unfolded states could play an important role in the aggregation of proteins during the long-term storage of aqueous protein formulations.

US$29.90

PMID: 14661914


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