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Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters

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Abstract

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

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Abbreviations

DOSY:

Diffusion ordered spectroscopy

DECRA:

Direct exponential curve resolution algorithm

PFGSE NMR:

Pulse field gradient spin echo NMR

3D HSQC-iDOSY:

Heteronuclear single quantum correlation-internal encoded diffusion ordered spectroscopy

Dav × 10−10 m2 s−1 :

Translational diffusion coefficient for assembly of oligomers

Di × 10−10 m2 s−1 :

Translational diffusion coefficient for individual oligomer

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Acknowledgements

This research was undertaken under the frame of National Centre of Research and Development PBS2/A27/09/2013. The authors gratefully acknowledge the reviewer for putting their attention to published work of Patil et al. (2017), which appeared nearly concurrently to the present submission.

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Authors and Affiliations

  1. National Medicines Institute, Chełmska 30, 00-725, Warsaw, Poland

    Jerzy Sitkowski, Wojciech Bocian, Elżbieta Bednarek & Lech Kozerski

  2. Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089, Warsaw, Poland

    Mateusz Urbańczyk & Wiktor Koźmiński

  3. Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516, Warsaw, Poland

    Piotr Borowicz, Grażyna Płucienniczak, Natalia Łukasiewicz & Iwona Sokołowska

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  1. Jerzy Sitkowski
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Correspondence to Lech Kozerski.

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Sitkowski, J., Bocian, W., Bednarek, E. et al. Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters. J Biomol NMR 71, 101–114 (2018). https://doi.org/10.1007/s10858-018-0197-y

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