Arginine dipeptides affect insulin aggregation in a pH‐ and ionic strength‐dependent manner

Abstract

Solutions containing arginine or mixtures of arginine and other amino acids are commonly used for protein liquid formulations to overcome problems such as high viscosities, aggregation, and phase separation. The aim of this work is to examine whether the stabilizing properties of arginine can be improved by incorporating the amino acid into a dipeptide. A series of arginine-containing dipeptides have been tested for their ability to suppress insulin aggregation over a range of pH and ionic strength. The aggregation is monitored at room temperature using a combination of turbidimetry and light scattering for solutions at pH 5.5 or 3.7, whereas thermal-induced aggregation is measured at pH 7.5. In addition, intrinsic fluorescence has been used to quantify additive binding to insulin. The dipeptide diArg is the most effective additive in solutions at pH 5.5 and 3.7, whereas the dipeptide Arg-Phe almost completely eliminates thermally-induced aggregation of insulin at pH 7.5 up to temperature of 90°C. Insulin has been chosen as a model system because the molecular forces controlling its aggregation are well known. From this understanding, we are able to provide a molecular basis for how the various dipeptides affect insulin aggregation.

Enhancing the crystallisation of insulin using amino acids as soft-templates to control nucleation

“…At this pH, arginine binds to the negatively charged residues on the insulin surface, such as glutamine or histidine, and neutralises repulsive electrostatic interaction, leading to an enhanced insulin aggregation and crystallisation. Enhanced insulin aggregation due to the addition of arginine at pH around 7.5 have already been reported in other studies (61). At higher arginine concentration (>0.06 M), the crystal occurrence starts to decrease (e.g.…”

“…However, arginine at higher ionic strengths and concentration also interacts with the dimer at the His10 position which could lead to a replacement of the Zn 2+ ions with the guanidinium group of the arginine residues (65), leading to the zinc free rhombic dodecahedral crystal shape. Furthermore, it has been shown that the surface free energy of insulin crystals with the rhombic dodecahedral shape is lower than insulin crystals with a cubic shape (61), which could indicate a change in the interfacial energy between the critical nucleus and the bulk solution with the addition of arginine. Or 2) The increase in pH stemming from the protonated arginine molecules in solution promotes the formation of hexagonal insulin crystals.…”

Structure-based discovery of a new protein-aggregation breaking excipient

“…The non-specific stabilization observed with a mAb and the non-stabilizing effect of compound A's enantiomer with IFN both strongly support our initial hypothesis of a specific proteinligand interaction leading to a stabilization against native protein aggregation of IFN. It is important to point out that the stabilizing effect of compound A may very well be pH dependent, especially due to its multiple titratable sites which could result in a pH dependent protein-ligand interaction profile (42).…”

Arginine and Arginine-Rich Peptides as Modulators of Protein Aggregation and Cytotoxicity Associated With Alzheimer’s Disease

“…Continued studies over the past decade, however, have demonstrated the effects of arginine on protein aggregation vary depending on its context and concentration, and are not always suppressive. Arginine potently inhibits the aggregation of lysozyme (Matsuoka et al, 2007;Ito et al, 2011), and porcine and mink growth hormones (Cirkovas and Sereikaite, 2011); monomeric arginine prevents the oligomerisation of insulin (Varughese and Newman, 2012;Březina et al, 2018;Haghighi-Poodeh et al, 2020), and di-arginine peptides exhibit even higher efficacy for suppressing insulin aggregation (Nuhu and Curtis, 2015). At physiological pH, arginine hydrochloride also suppresses the aggregation of immunoglobulin G1 (IgG1), with this effect attributed to the interactions between arginine and hydrophobic IgG1 residues (Fukuda et al, 2014).…”