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Cited by 132Protein purification and stabilityProtein Interaction Studies and Fluorescence AnalysisAnalytical Methods in Pharmaceuticals

UV-Light Exposure of Insulin: Pharmaceutical Implications upon Covalent Insulin Dityrosine Dimerization and Disulphide Bond Photolysis

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Correia, Manuel, Neves-Petersen, Maria Teresa, Jeppesen, Per Bendix, Gregersen, Søren, Petersen, Steffen B.·2012·PLoS ONE
DOI 10.1371/journal.pone.0050733PMC PMC3515625
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Abstract

In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2)) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein structure. Structural damage includes insulin dimerization via dityrosine cross-linking or disulphide bond disruption, which affects the hormone's structure and bioactivity.

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  • Tyrosine is redox active; its phenolic side-chain is easily oxidized because the intermediary tyrosyl radical is stabilized by mesomeric delocalization.

    “In particular, tyrosine is a redox active structure. The phenolic side-chain is easily oxidized, because the intermediary tyrosyl radical is stabilized by mesomeric delocalization of the unpaired electron (Fig. 2A) [22].”

    From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [22]

    The Introduction (page unavailable) describes electron loss from excited tyrosine followed by formation of a tyrosyl radical, and the Discussion (page unavailable) calls that radical relatively stable. These passages support tyrosine's redox activity, but do not say that its phenolic side-chain is easily oxidized or attribute radical stability to mesomeric delocalization. (LLM, conf=0.84)

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Cited by

Sorted by citations

Authors
Correia, M., Neves-Petersen, M. T., Jeppesen, P. B., Gregersen, S., & Petersen, S. B.
Affiliations
  • Aalborg University (DK)
  • Aarhus University Hospital (DK)
  • International Iberian Nanotechnology Laboratory (PT)
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-12-05
Volume
7
Issue
12
Pages
e50733
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0050733
PMC
PMC3515625
OpenAlex
W2007514795
Cited by
132
References
1
Citations by year
2026: 11 · 2025: 12 · 2024: 8 · 2023: 11 · 2022: 7 · 2021: 14 · 2020: 14 · 2019: 13 · 2018: 9 · 2017: 10 · 2016: 8 · 2015: 8 · 2014: 5 · 2013: 2
FWCI
1.93
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • VBN Forskningsportal (Aalborg Universitet) (submittedVersion)
Funders
  • Fundação para a Ciência e a Tecnologia (SFRH/BD/61012/2009, /2009)
  • European Social Fund
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Interaction Studies and Fluorescence Analysis
  • Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
Keywords
human insulin, UV exposure, dityrosine cross-linking, glucose uptake, drug stability, pharmaceutical quality control
MeSH terms
Absorption, Animals, Binding Sites, Circular Dichroism, Cross-Linking Reagents, Disulfides, Glucose, Guinea Pigs, Humans, Insulin, Kinetics, Photolysis, Radioimmunoassay, Spectrometry, Fluorescence, Sulfhydryl Compounds, Tyrosine, Ultraviolet Rays, Protein Structure, Tertiary, Dimerization

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