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

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

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

Noncovalent PEGylation through Protein–Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation

Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein–polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-<i>block</i>-poly­(<i>N</i>,<i>N</i>-dimethylaminoethyl) (PEG-<i>b</i>-PAMA) inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of α-amylase activity by PEG-<i>b</i>-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-<i>b</i>-PAMA molecule was initially located at six different placements of the <i>x</i>-, <i>y</i>-, and <i>z</i>-axis ±20 Å from the center of α-amylase, which showed that the PEG-<i>b</i>-PAMA nonspecifically bound to the α-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-<i>b</i>-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-<i>b</i>-PAMA did not interact with α-amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte

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

One-Step Identification of Antibody Degradation Pathways Using Fluorescence Signatures Generated by Cross-Reactive DNA-Based Arrays

Therapeutic antibodies are prone to degradation via a variety of pathways during each stage of the manufacturing process. Hence, a low-cost, rapid, and broadly applicable tool that is able to identify when and how antibodies degrade would be highly desirable to control the quality of therapeutic antibody products. With this goal in mind, we have developed signature-based sensing system to discriminate differently degraded therapeutic antibodies. The use of arrays consisting of conjugates between nanographene oxide and fluorophore-modified single-stranded DNAs under acidic pH conditions generated unique fluorescence signatures for each state of the antibodies. Multivariate analyses of the thus obtained signatures allowed identifying (i) common features of native, denatured, and visibly aggregated antibodies, (ii) complicated degradation pathways of therapeutic omalizumab upon time-course heat-treatment, and (iii) the individual compositions of differently degraded omalizumab mixtures. As the signature-based sensing has the potential to identify a broad range of degraded antibodies formed by different kinds of realistic stress types, this system may serve as the basis for high-throughput assays for the screening of antibody manufacturing processes