Interferon-β (INF-β) Characterization

Introduction to Interferon-β (INF-β)

Interferon-β (INF-β) is part of a protein group collectively referred to as interferons (IFNs). Historically, the classification system for IFNs was grounded in the determination of their cellular origins, meaning the primary three divisions were leukocyte-, fibroblast-, and immune-interferons corresponding to their synthesis primarily in leukocytes, fibroblasts, and T-lymphocytes, respectively.

Fig 1. Interferon protein structures. (N., D., & D., B. 2012)

However, as our comprehension of INF structure and functionality has advanced, so has the nomenclature of IFN. Presently, the three prime categories of INF are labelled as INF-α, INF-β, and INF-γ. Between human INF-α and INF-β, there is an approximate 30% similarity at the primary amino acid sequence level. Conversely, INF-γ shares no marked similarity with either.

Structure of INF-β

INF-β is endowed with three cysteine residues that are situated at the 17th, 31st, and 141st positions of the amino acid sequence. It is plausible that these cysteine residues partake in intermolecular disulfide bridging, thereby leading to the creation of inactive dimers and oligomers. Concurrently, a random interaction within each molecule could occur among the three cysteines, leading to the presence of three different molecular species within the cell.

Fig 2. Schematic representation of the crystallographic dimer of huIFN-β. (Karpusas, M., et al. 1997)

Each of these possesses one of the three potentially possible intramolecular disulfide bridges. It has been conjectured that only one is likely to mimic the native conformation and hence maintain biological viability. Both these scenarios could collectively contribute to the creation of inactive monomers and oligomers within the cell. If in fact the sulfhydryls were accountable for the diminished specific activity of the INF-β protein, then the exclusion of one of the cysteines could make way for the sole unique intramolecular disulfide bridge formation. Thereby, subsequently limiting the formation of free-sulfhydryl groups that can facilitate the generation of dimers. or oligomers.

Stability of INF-β

The stability of INF-β has been found to be associated with its formulation parameters. A non-carrier protein solution formulation of INF-β, with a per milliliter composition of 1.2 mg INF-β, 10 mg of SDS in a 50 mM sodium acetate base and 2 mM EDTA, and a pH of 5.5, was used in the experiments. We subjected the collected data to an Arrhenius fit. The resultant t90 value, indicative of the time taken to achieve 90% purity of Interferon, derived from the SDS-PAGE and RP-HPLC data, was predicted to be around 7 years at 5°C, within a range of 2–8°C. Moreover, the rate of INF-β degradation was observed to have an activation energy of 24 kcal/mole.

Fig 3. Potency stability of INF-β at 5°C. (Pearlman, Rodney, and Y. John Wang, eds. 1996)

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References

1. N., D., & D., B. Interferon and Apoptosis in Systemic Lupus Erythematosus. Systemic Lupus Erythematosus. 2012.
2. Karpusas, M., et al. The crystal structure of human interferon at 2.2-A resolution. Proceedings of the National Academy of Sciences. 1997, 94(22), 11813–11818.
3. Pearlman, Rodney, and Y. John Wang, eds. Formulation, characterization, and stability of protein drugs. Vol. 9. Springer Science & Business Media. 1996.

For research use only, not intended for any clinical use.