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Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion
Published on: May 10, 2018
Analysis of insulin allostery in solution and solid state with FTIR
Morten Jonas Maltesen1, Simon Bjerregaard, Lars Hovgaard
1Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. mjm@farma.ku.dk
Journal of Pharmaceutical Sciences
|April 3, 2009
Summary
Fourier Transform Infrared (FTIR) spectroscopy effectively monitors the T-R transition in insulin hexamers upon phenol binding. This method supports maintaining the allosteric state in solid-state insulin samples.
Area of Science:
- Biochemistry
- Structural Biology
- Spectroscopy
Background:
- The insulin hexamer exists in equilibrium between T(6), R(3)T(3), and R(6) states.
- Phenol binding stabilizes the R(6) state by interacting with specific hydrophobic pockets.
- Conformational changes, particularly in B1-B8 residues (extended to alpha-helix), differentiate T(6) and R(6) states.
Purpose of the Study:
- To evaluate Fourier Transform Infrared (FTIR) spectroscopy for monitoring the T-R transition in insulin hexamers induced by phenol.
- To explore FTIR's utility for analyzing solid-state insulin samples.
- To determine if the allosteric state can be preserved in dried insulin.
Main Methods:
- Solution-state FTIR spectroscopy of insulin with and without phenol.
- Circular Dichroism (CD) spectroscopy for comparison.
- Lyophilization of insulin samples.
- FTIR analysis of lyophilized and reconstituted samples.
- Principal Component Analysis (PCA) of FTIR spectra.
Main Results:
- FTIR spectra showed an increase in alpha-helix content upon phenol binding in solution, correlating with CD data.
- Dissociation constants derived from FTIR and CD were comparable.
- The increased alpha-helix content was retained after lyophilization.
- Lyophilization altered overall FTIR spectra, but the alpha-helix increase persisted.
- Reconstitution restored the structure to a state similar to pre-lyophilization.
Conclusions:
- FTIR spectroscopy is a viable alternative to CD for monitoring insulin's T-R transition.
- FTIR enables the study of insulin's allosteric states in solid-state and lyophilized forms.
- The allosteric conformational changes in insulin hexamers can be preserved post-lyophilization.
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