Relationships between protein adsorption and isoelectric point (A) or hydrophobicity (B) of proteins.

<p>The solutions containing 0.1 mg/mL proteins and 58 m²/L PS particles in 10 mM Na-phosphate buffer at pH 7.0 was incubated at 25°C for 1 h. After centrifugation, protein concentrations in the supernatant were determined.</p

Effects of additives on lysozyme adsorption monitored by the concentration (A) and activity (B).

<p>The solutions containing lysozyme and additives were mixed with PS particles in 10 mM Na-phosphate buffer at pH 7.0, and then incubated at 25°C for 1 h. After centrifugation, protein concentration (A) and enzyme activity (B) in the supernatant were determined. The final concentrations of lysozyme and PS particles were 0.1 mg/mL and 58 m²/L, respectively.</p

Effects of additives on lysozyme adsorption when the order of mixing was changed.

<p>The annotation “Desorption” (white bars) corresponds to the case where the additives were added after adsorption of lysozyme on the PS particles. The final concentrations of lysozyme, PS particles, and additives were 0.1 mg/mL, 58 m²/L, and 500 mM, respectively. The annotation “Adsorption” (gray bars) means that the additives were mixed with lysozyme before the addition of PS particles. The final concentrations of lysozyme, PS particles, and additives were 0.067 mg/mL, 38 m²/L, and 500 mM, respectively. After centrifugation of each sample, protein concentration (A) and enzyme activity (B) in the supernatant were determined.</p

Time course of protein desorption (A) and enzyme activity (B) of lysozyme in the supernatant.

<p>The additives were added after adsorption of lysozyme on the PS particles. The final concentrations of lysozyme, PS particles, and additives were 0.067 mg/mL, 38 m²/L, and 500 mM, respectively.</p

Effects of additives on ChT adsorption monitored by concentration (A) and activity (B).

<p>The solutions containing ChT and additives were mixed with PS particles in 10 mM Na-phosphate buffer at pH 7.0, and then incubated at 25°C for 1 h. After centrifugation, protein concentration (A) and enzyme activity (B) in the supernatant were determined. The final concentrations of ChT and PS particles were 0.1 mg/mL and 58 m²/L, respectively.</p

Effects of additives on protein adsorption as a function of protein hydrophobicity.

<p>The solutions containing proteins and 500 mM additives were mixed with PS particles in 10 mM Na-phosphate buffer at pH 7.0, and then incubated at 25°C for 1 h. After centrifugation, protein concentration in the supernatant was determined. The concentration of proteins and PS particles were as follows: (i) Lysozyme, ChT, and BSA–0.1 mg/mL protein and 58 m²/L PS particles; (ii) RNase A–0.1 mg/mL protein and 116 m²/L PS particles; (iii) Subtilisin–0.25 mg/mL protein and 58 m²/L PS particles.</p

Molecular Dynamics Simulation of the Arginine-Assisted Solubilization of Caffeic Acid: Intervention in the Interaction

We have previously demonstrated that arginine increases the solubility of aromatic compounds that have poor water solubility, an effect referred to as the “arginine-assisted solubilization system (AASS)”. In the current study, we utilized a molecular dynamics simulation to examine the solubilization effects of arginine on caffeic acid, which has a tendency to aggregate in aqueous solution. Caffeic acid has a hydrophobic moiety containing a π-conjugated system that includes an aromatic ring and a hydrophilic moiety with hydroxyl groups and a carboxyl group. While its solubility increases at higher pH values due to the acquisition of a negative charge, the solubility was greatly enhanced by the addition of 1 M arginine hydrochloride at any pH. The results of the simulation indicated that the caffeic acid aggregates were dissociated by the arginine hydrochloride, which is consistent with the experimental data. The binding free energy calculation for two caffeic acid molecules in an aqueous 1 M arginine hydrochloride solution indicated that arginine stabilized the dissociated state due to the interaction between its guanidinium group and the π-conjugated system of the caffeic acid. The binding free energy of two caffeic acid molecules in the arginine hydrochloride solution exhibited a local minimum at approximately 8 Å, at which the arginine intervened between the caffeic acid molecules, causing a stabilization of the dissociated state of caffeic acid. Such stabilization by arginine likely led to the caffeic acid solubilization, as observed in both the experiment and the MD simulation. The results reported in this paper suggest that AASS can be attributed to the stabilization resulting from the intervention of arginine in the interaction between the aromatic compounds

Molecular Dynamics Simulation of the Arginine-Assisted Solubilization of Caffeic Acid: Intervention in the Interaction

We have previously demonstrated that arginine increases the solubility of aromatic compounds that have poor water solubility, an effect referred to as the “arginine-assisted solubilization system (AASS)”. In the current study, we utilized a molecular dynamics simulation to examine the solubilization effects of arginine on caffeic acid, which has a tendency to aggregate in aqueous solution. Caffeic acid has a hydrophobic moiety containing a π-conjugated system that includes an aromatic ring and a hydrophilic moiety with hydroxyl groups and a carboxyl group. While its solubility increases at higher pH values due to the acquisition of a negative charge, the solubility was greatly enhanced by the addition of 1 M arginine hydrochloride at any pH. The results of the simulation indicated that the caffeic acid aggregates were dissociated by the arginine hydrochloride, which is consistent with the experimental data. The binding free energy calculation for two caffeic acid molecules in an aqueous 1 M arginine hydrochloride solution indicated that arginine stabilized the dissociated state due to the interaction between its guanidinium group and the π-conjugated system of the caffeic acid. The binding free energy of two caffeic acid molecules in the arginine hydrochloride solution exhibited a local minimum at approximately 8 Å, at which the arginine intervened between the caffeic acid molecules, causing a stabilization of the dissociated state of caffeic acid. Such stabilization by arginine likely led to the caffeic acid solubilization, as observed in both the experiment and the MD simulation. The results reported in this paper suggest that AASS can be attributed to the stabilization resulting from the intervention of arginine in the interaction between the aromatic compounds