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Related Experiment Videos

Acid stabilization of insulin

C Bryant1, D B Spencer, A Miller

  • 1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.

Biochemistry
|August 17, 1993
PubMed
Summary

Acidification surprisingly stabilizes insulin's structure by increasing unfolding energy. This effect, linked to histidine protonation, is crucial for insulin's stability within pancreatic beta-cell granules.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Chemistry

Background:

  • Insulin is a crucial hormone for glucose regulation.
  • Understanding insulin's conformational stability is vital for its therapeutic use and biological function.
  • The stability of proteins can be significantly influenced by environmental factors like pH.

Purpose of the Study:

  • To investigate the effect of pH on the conformational stability of insulin.
  • To elucidate the molecular mechanisms underlying pH-dependent insulin stability.
  • To explore the physiological relevance of insulin's acid stabilization.

Main Methods:

  • Studied insulin's conformational stability using Gibbs free energy of unfolding measurements.
  • Performed pH titration to determine the pK(a) of the stability transition.

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  • Utilized 1H-NMR spectroscopy to analyze histidine protonation.
  • Employed selective amino acid alteration and measured enthalpies of ionization.

    • Main Results:

    • Acidification increased insulin's Gibbs free energy of unfolding by approximately 30%.
    • A single proton transition with an apparent pK(a) of 7.0 described the pH-dependent stability.
    • Acid stabilization was attributed to the protonation of histidine at position 5 on the B-chain (HB5).
    • Near-UV circular dichroism indicated a conformational change in tertiary structure upon stabilization.

    Conclusions:

    • Insulin exhibits significant acid stabilization of its conformation.
    • Protonation of HB5 is the key mechanism for this acid stabilization.
    • This phenomenon is likely physiologically important for maintaining insulin stability in pancreatic beta-cell secretory/storage granules.