Characterization of Sizes of Aggregates of Insulin Analogs and the Conformations of the Constituent Protein Molecules: A Concomitant Dynamic Light Scattering and Raman Spectroscopy Study

Abstract

To generate aggregates, 3 insulin analogs, lispro, aspart, and glulisine, were incubated without phenolic preservatives for 30 days at 37 °C. As a function of incubation time, aggregation was quantified with size exclusion chromatography, and the sizes of aggregates and the conformations of the constituent molecules were characterized with concomitant dynamic light scattering and Raman spectroscopy. During incubation, lispro was progressively converted into soluble aggregates with hydrodynamic diameters of circa 15 nm, and 95% of the native protein had aggregated at day 30. Raman spectroscopy documented that aggregation resulted in conversion of a large fraction of native alpha helix into nonnative beta sheet structure and a distortion of disulfide bonds. In contrast, for aspart and glulisine only 20% of the native proteins aggregated after 30 days, and minimal structural perturbations were detected. In addition, consistent with the relative aggregation rates during isothermal incubation, Raman spectroscopy showed that during heating the onset temperature for secondary structural perturbations of lispro occurred 7 °C-10 °C lower than those for aspart or glulisine. Overall the results of this study demonstrated that-as in the case during formation of amyloid fibrils from insulin-formation of soluble aggregates of lispro resulted in a high level of conversion of alpha helix into beta sheet.

An Evaluation of the Delivery of Medicines Using Drones

“…Apart from the formation of large fibrils, insulin solutions are also known to form much smaller aggregates in the nm region [26], an order of magnitude smaller than the formazin particles [36,37]. DLS was used to complement the UV turbidity analysis as it is a sensitive method for measuring such small-sized aggregates, 1 to 450 nm in diameter [38,39]. The time-dependent fluctuations in the scattering intensity as a result of Brownian motion are measured by DLS, the frequency of these fluctuations are used to determine particle diffusion coefficients and particle size [38].…”

Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2

“…The second peak at ~100 nm can be assigned to small soluble rhBMP-2 nanoparticles. The height or area of both peaks cannot be used for particle quantification as light scattering intensity increases proportional to the power of six with the particle diameter and thus larger particles appear overrepresented [33]. For estimating the relative level of different size populations the volume distribution is more suitable.…”

Determination of amyloid core regions of insulin analogues fibrils

“…Unfortunately, often the experimental conditions differ significantly from different authors, which creates certain difficulties in interpreting the results. In another study, Zhou et al showed that lispro aggregates faster than glulisine and aspart without stirring at the same temperature of 37°C, but different buffer conditions were used for each analogue [63]. Our data show that the insulin analogues form fibrils more slowly than insulin.…”