Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Abstract

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat-induced deamidation and β-elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alcohol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S-carboxy-methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers--PEG and PVP--and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution.

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

“…The same trend was observed when the nanogel concentration was increased (Figure S5). In the presence of NG-F, less than 10% fibrillation is observed even after heating at high temperatures for 30 minutes, a value that is much lower than that obtained with linear polymers (poly-SPB) and previously reported compounds such as non-detergent sulfobetaines3334353637 and L-arginine hydrochloride3839 (Figure S6). …”

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

“…This effect can prevent alteration of protein's conformation and aggregation upon water removal from the protein surface during freezing [15]. Furthermore, arginine is one of the most commonly used additive for prevention of noncovalent protein aggregation [28].…”

Oligoethylene glycols prevent thermal aggregation of α‐chymotrypsin in a temperature‐dependent manner: Implications for design guidelines

“…Protein aggregation involves physical and chemical processes; the former is often caused by non‐covalent hydrophobic interactions, while the latter is responsible for covalent cross‐link formation, such as disulfide exchange reaction. It is likely that oligoethylene glycols are effective to prevent protein aggregation in the former cases, whereas the other solution additives are required for the latter case, as suggested previously …”

“…PEG generally accelerates protein‐protein associations at room temperature due to its excluded volume effect . In contrast, PEG prevents protein–protein interactions at high temperature . This temperature dependence was probably attributable to the increasing hydrophobic interactions at higher temperatures.…”

“…Water‐soluble and amphiphilic poly(ethylene glycol) (PEG) is considered a candidate additive for the suppression of heat‐induced aggregation of proteins, because PEG tends to interact with hydrophobic side chains on proteins, particularly at elevated temperatures . In fact, PEG inhibits interactions between denatured proteins, resulting in increased refolding yield and prevention of thermal inactivation of proteins . However, high molecular weight PEG is not an adequate solution additive due to its high viscosity and difficulty of removal from protein solution.…”