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Chemical Modification of the Amino Groups of Human Insulin: Investigating Structural Properties and Amorphous Aggregation of Acetylated Species

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Abstract

The efficacy of human recombinant insulin can be affected by its aggregation. Effects of acetylation were observed on insulin structure, stability, and aggregation at 37 and 50 °C and pH of 5.0 and 7.4 with the use of spectroscopy, circular dichroism (CD), dynamic light scattering (DLS), and atomic force microscopy (AFM). Raman and FTIR results were indicative of structural changes in AC-INS, and CD analyses showed a slight increase in β-sheet content in AC-INS. Melting temperature (Tm) measurements indicated an overall more stable structure and spectroscopic assessment showed a more compact one. Formation of amorphous aggregates was followed over time and kinetics parameters showed a longer nucleation phase (higher t* amount) and lower aggregates amount (lower Alim) for acetylated insulin (AC-INS) compared to native (N-INS) in all tested conditions. The results of amyloid-specific probes approved the formation of amorphous aggregates. Size particle and microscopic analysis suggested that AC-INS was less prone to form aggregates, which were smaller if formed. In conclusion, this study has demonstrated that controlled acetylation of insulin may lead to its higher stability and lower propensity toward amorphous aggregation and has provided insight into the result of this type of post-translational protein modification.

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Abbreviations

INS:

Insulin

N-INS:

Native Insulin

AC-INS:

Acetylated Insulin

HI-INS:

Heat-Induced-INS

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Acknowledgements

The support of the Institute of Biochemistry and Biophysics of the University of Tehran, Iran, is gratefully acknowledged.

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Authors and Affiliations

  1. Laboratory of Biophysics and Molecular Biology, Departments of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., P.O. Box 13145-1384, Tehran, Iran

    Reyhane Kamelnia, Bahram Goliaei & Seyed Peyman Shariatpanahi

  2. Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran

    Faramarz Mehrnejad

  3. Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

    Atiyeh Ghasemi & Ashkan Zare Karizak

  4. Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Jalal Al Ahmad Highway, Tehran, 1411713137, Iran

    Azadeh Ebrahim-Habibi

  5. Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

    Azadeh Ebrahim-Habibi

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  1. Reyhane Kamelnia
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Contributions

This work reports part of RK project thesis to which BG and AEH have been supervisors and FM has been an advisor. The project was defined and discussed upon by BG, AEH, FM and RK, followed by supervisions, data checking and discussions by the supervisors and advisor as well as SPS. RK did the main body of the experiments, and AG and AZK have taken part into the research experiments and data analysis. First draft has been prepared by RK with help from AZK and revised by SPS. All authors have taken part in further revising and finalizing the manuscript.

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Correspondence to Bahram Goliaei or Azadeh Ebrahim-Habibi.

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Kamelnia, R., Goliaei, B., Peyman Shariatpanahi, S. et al. Chemical Modification of the Amino Groups of Human Insulin: Investigating Structural Properties and Amorphous Aggregation of Acetylated Species. Protein J 42, 383–398 (2023). https://doi.org/10.1007/s10930-023-10131-7

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