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Pharmaceutical Research

, 35:137 | Cite as

Denaturation and Aggregation of Interferon-τ in Aqueous Solution

  • Ryan R. Manning
  • Glenn A. Wilson
  • Ryan E. Holcomb
  • Nathaniel J. Zbacnik
  • Auria A. Tellechea
  • Chelsey L. Gilley-Dunn
  • Ryan J. Krammes
  • Nathan S. Krammes
  • Gabriel J. Evans
  • Charles S. Henry
  • Mark Cornell Manning
  • Brian M. Murphy
  • Robert W. Payne
  • Derrick S. KatayamaEmail author
Research Paper
  • 193 Downloads

Abstract

Purpose

To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.

Methods

Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system.

Results

The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1.

Conclusion

Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Key words

AF4 aggregation chaotropes denaturation flow microscopy guanidinium hydrochloride interferon-tau protein conformational stability urea 

Abbreviations

ΔG

Gibbs free energy of unfolding

AF4

Asymmetrical flow field-flow fractionation

DLS

Dynamic light scattering

GnHCl

Guanidinium hydrochloride

IFN-τ

Interferon-τ

MALLS

Multi-angle laser light scattering

SEC

Size exclusion chromatography

UV

Ultraviolet

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ryan R. Manning
    • 1
  • Glenn A. Wilson
    • 2
  • Ryan E. Holcomb
    • 3
    • 4
  • Nathaniel J. Zbacnik
    • 3
  • Auria A. Tellechea
    • 3
    • 4
  • Chelsey L. Gilley-Dunn
    • 3
    • 4
  • Ryan J. Krammes
    • 3
  • Nathan S. Krammes
    • 3
  • Gabriel J. Evans
    • 3
  • Charles S. Henry
    • 4
  • Mark Cornell Manning
    • 3
    • 4
  • Brian M. Murphy
    • 5
  • Robert W. Payne
    • 3
    • 4
  • Derrick S. Katayama
    • 3
    • 4
    Email author
  1. 1.Great Lakes Bio DesignCharlotteUSA
  2. 2.West Coast BioDesignSanta BarbaraUSA
  3. 3.Legacy BioDesign LLCJohnstownUSA
  4. 4.Department of ChemistryColorado State UniversityFort CollinsUSA
  5. 5.Alder BiopharmaceuticalsSeattleUSA

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