Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols

Abstract

Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2-7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin's native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30-35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in α-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular.

Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

“…Ethanol preferentially binds to the protein, modifies the surrounding hydration layer, and increases the repulsion between molecules. This results in a delay of the aggregation process, which is observed as a prolonged lag-phase in the kinetics [40,41]. A similar effect was observed, when comparing the kinetics for AA spherulites and EtOH spherulites (Fig.…”

The Possible Role of the Type I Chaperonins in Human Insulin Self-Association

“…We next examined the morphologies of the insulin aggregates obtained in the presence and in the absence (control) of the chaperonins by AFM ( Figure 7 ). As expected for insulin aggregated at neutral pH [ 21 , 40 ], fibrillary material tends to form short–stumpy fibril clusters ( Figure 7 a), with a different morphology than elongated fibrils typically observed for insulin amyloid species formed at pH 2.2 [ 14 ]. In contrast, a granular species dense network is present in the image from the protein sample incubated with the chaperonins.…”

“…Several observations showed how the presence of higher-order oligomeric states in the initial insulin sample is at the basis of a self-inhibition process that determines an unexpected dependence of amyloid aggregation reaction half-times (time at which ThT fluorescence reaches the half-maximum value) on the insulin concentration monomers [ 58 ]. Hence, according to the classical nucleation-polymerisation model, whereas at an acidic pH, the insulin sample, which is essentially populated by monomers/dimers, aggregates with a rate that increases with the concentration, at a neutral pH in which higher-order oligomeric states are predominant, the amyloid formation has an opposite behaviour at varying concentrations [ 21 ]. Probably the higher-order oligomeric states are amyloid off-pathway conformations that do not participate in fibril elongation (thus reducing the low molecular weight species prone to aggregate) or interfere with it.…”

“…Several studies in vitro contributed to understanding the molecular basis of the insulin amyloid self-assembly process and clarifying the role of environmental factors in the insulin fibril formation kinetics [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. In particular, both insulin aggregation rates and fibril morphologies are strongly influenced by the solution pH; at an acidic pH, the process starts from a homogeneous monomer/dimer population, while at increasing pH values, the equilibrium shifts towards higher molecular weight oligomers, whose dissociation may be rate limiting or partially rate limiting [ 20 , 21 ].…”

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements

“…By measuring the kinetics of a particular physical parameter of the system, one may obtain the fibrillation kinetics described by a sigmoid curve in most cases. The conventional approach is to determine several parameters from the aggregation kinetic curve such as the duration of the lag phase and the growth rate and to investigate their behavior upon changing parameters of the system, both molecular environment (ionic strength, temperature, protein concentration, the presence of inhibitory molecules) and molecular structure (e.g., by inducing mutations in the protein structure) in order to study the fibrillation process [ 5 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ].…”