Kentaro Shiraki

Effect of Additives on Protein Aggregation

This paper overviews solution additives that affect protein stability and aggregation during refolding, heating, and freezing processes. Solution additives are mainly grouped into two classes, i.e., protein denaturants and stabilizers. The former includes guanidine, urea, strong ionic detergents, and certain chaotropic salts; the latter includes certain amino acids, sugars, polyhydric alcohols, osmolytes, and kosmotropic salts. However, there are solution additives that are not unambiguously placed into these two classes, including arginine, certain divalent cation salts (e.g., MgCl(2)) and certain polyhydric alcohols (e.g., ethylene glycol). Certain non-ionic or non-detergent surfactants, ionic liquids, amino acid derivatives, polyamines, and certain amphiphilic polymers may belong to this class. They have marginal effects on protein structure and stability, but are able to disrupt protein interactions. Information on additives that do not catalyze chemical reactions nor affect protein functions helps us to design protein solutions for increased stability or reduced aggregation.

Arginine Increases the Solubility of Coumarin: Comparison with Salting-in and Salting-out Additives

Poor aqueous solubility of low molecular weight drug substances hampers their development as pharmacological agents. Here, we have examined the effects of arginine on the solubility of organic compounds, coumarin, caffeine and benzyl alcohol, in aqueous solution. Arginine increased the solubility of aromatic coumarin, but not non-aromatic caffeine, concentration dependently, suggesting the favourable interaction of arginine with the aromatic structure. Consistent with this, arginine also increased the solubility of aromatic benzyl alcohol. Guanidine hydrochloride, urea and salting-in salts increased both coumarin and caffeine solubilities, while salting-out salts decreased them. These results suggest the specific interaction of arginine with aromatic groups, leading to increased solubility of coumarin. However, the effect of 1 M arginine on coumarin solubility was at most approximately 2-fold, which may limit its applications as a solubility enhancing agent.

High-resolution X-ray analysis reveals binding of arginine to aromatic residues of lysozyme surface: implication of suppression of protein aggregation by arginine

While biotechnological applications of arginine (Arg) as a solution additive that prevents protein aggregation are increasing, the molecular mechanism of its effects remains unclear. In this study, we investigated the Arg-lysozyme complex by high-resolution crystallographic analysis. Three Arg molecules were observed to be in close proximity to aromatic amino acid residues of the protein surface, and their occupancies gradually increased with increasing Arg concentration. These interactions were mediated by electrostatic, hydrophobic and cation-π interactions with the surface residues. The binding of Arg decreased the accessible surface area of aromatic residues by 40%, but increased that of charged residues by 10%. These changes might prevent intermolecular hydrophobic interactions by shielding hydrophobic regions of the lysozyme surface, resulting in an increase in protein solubility.

Discovery of posttranslational maturation by self-subunit swapping

Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of β- and α-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobalt-containing L-NHase (holo-L-NHase containing Cys-SO2− and Cys-SO− metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt- and oxidized cysteine-containing α-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free α-subunit of apo-L-NHase with the cobalt-containing cysteine-modified α-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named “self-subunit swapping.”

Arginine-assisted solubilization system for drug substances: solubility experiment and simulation.

The poor aqueous solubility of drug substances hampers their broader applications. This paper describes a de novo strategy to increase the aqueous solubility of drug substances using an arginine-assisted solubilization system (AASS) with alkyl gallates as model drug substances. Solubility experiments of alkyl gallates showed that arginine greatly increases the aqueous solubility of different alkyl gallates, whose aqueous solubilities differ widely. In contrast, lysine showed marginal effects on alkyl gallates solubility. Molecular dynamic simulation indicated a greater interaction of arginine with alkyl gallates than that of lysine, which reflects favorable interaction between the guanidinium group of arginine and the aromatic ring of alkyl gallates. Such interaction apparently disrupts association of alkyl gallate molecules, leading to solubilization. These results indicate AASS as a promising approach to solubilize poorly soluble drug substances containing aromatic ring structures.

Chemical modification of amino acids by atmospheric-pressure cold plasma in aqueous solution

Plasma medicine is an attractive new research area, but the principles of plasma modification of biomolecules in aqueous solution remain elusive. In this study, we investigated the chemical effects of atmospheric-pressure cold plasma on 20 naturally occurring amino acids in aqueous solution. High-resolution mass spectrometry revealed that chemical modifications of 14 amino acids were observed after plasma treatment: (i) hydroxylation and nitration of aromatic rings in tyrosine, phenylalanine and tryptophan; (ii) sulfonation and disulfide linkage formation of thiol groups in cysteine; (iii) sulfoxidation of methionine and (iv) amidation and ring-opening of five-membered rings in histidine and proline. A competitive reaction experiment using 20 amino acids demonstrated that sulfur-containing and aromatic amino acids were preferentially decreased by the plasma treatment. These data provide fundamental information for elucidating the mechanism of protein inactivation for biomedical plasma applications.

Specific Decrease in Solution Viscosity of Antibodies by Arginine for Therapeutic Formulations

Unacceptably high viscosity is observed in high protein concentration formulations due to extremely large therapeutic dose of antibodies and volume restriction of subcutaneous route of administration. Here, we show that a protein aggregation suppressor, arginine hydrochloride (ArgHCl), specifically decreases viscosity of antibody formulations. The viscosities of bovine gamma globulin (BGG) solution at 250 mg/mL and human gamma globulin (HGG) solution at 292 mg/mL at a physiological pH were too high for subcutaneous injections, but decreased to an acceptable level (below 50 cP) in the presence of 1,000 mM ArgHCl. ArgHCl also decreased the viscosity of BGG solution at acidic and alkaline pHs. Interestingly, ArgHCl decreased the viscosity of antibody solutions (BGG, HGG, and human immunoglobulin G) but not globular protein solutions (α-amylase and α-chymotrypsin). These results indicate not only high potency of ArgHCl as an excipient to decrease the solution viscosity of high concentration antibodies formulations but also specific interactions between ArgHCl and antibodies.

Regulation of lysozyme activity based on thermotolerant protein/smart polymer complex formation.

Proteins have evolved to acquire highly specialized biological functions and are ideal for various applications in both medicine and biotechnology, although denaturation is one of the major problems in protein chemistry. Here, we show a novel strategy for the regulation and preservation of the enzymatic activity even after heat treatment by the complex formation with a cationic smart copolymer, poly(N,N-diethylaminoethyl methacrylate)-graft-poly(ethylene glycol) (PEAMA-g-PEG). PEAMA-g-PEG suppressed the enzymatic activity of lysozyme completely without any conformational change, indicating complex formation and the capping of the active site of lysozyme by PEAMA-g-PEG. The addition of an anionic polymer, poly(acrylic acid) (PAAc), recovered the inhibited enzymatic activity of the lysozyme/PEAMA-g-PEG complex completely. Surprisingly, even after heating the lysozyme with PEAMA-g-PEG for 20 min at 98 degrees C, the addition of PAAc recovered 80% enzymatic activity of lysozyme. Circular dichroism (CD) spectral analysis clearly indicated that the irreversible inactivation of lysozyme induced by the heat treatment was suppressed by the complex formation with PEAMA-g-PEG.

Amino Acid Esters Prevent Thermal Inactivation and Aggregation of Lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat‐induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 °C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na‐phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 °C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat‐induced aggregation.

Indispensable structure of solution additives to prevent inactivation of lysozyme for heating and refolding

This article investigates solution additives that prevent misfolding of lysozyme from heating treatment and during refolding processes. Comparison of heat treatment of native lysozyme and oxidative refolding from the reduced and denatured state of lysozyme in the presence of 44 different additives revealed an indispensable chemical structure for the additives to be effective against heat‐induced misfolding and for refolding. The additives effective against heat treatment of native lysozyme possessed a main chain of the amino acid moiety. Amino acids that have esterificated and amidated carboxy groups prevented heat‐induced misfoldings more effectively than amino acids themselves. On the other hand, the additives effective against oxidative refolding possessed a guanidium or ureido group. The former additives prevented hydrophobic interaction between the main chains of the unfolded polypeptide, while the latter additives increased the solubility of the aromatic and aliphatic side‐chains. These data also support the fact that arginine (Arg) and Arg derivatives are versatile additives for both misfolding processes.