Noriko Yoshimoto

Calcein permeation across phosphatidylcholine bilayer membrane: effects of membrane fluidity, liposome size, and immobilization.

The permeation of calcein across the phospholipid bilayer membrane is a key phenomenon in the detection system using liposomes as a sensor unit. The behavior of the calcein release from the liposome was analyzed by a first-order kinetic to obtain the permeability coefficient, Ps [cm/s]. The Ps value for the neutral liposome, prepared by zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was found to depend on both the diameter of liposome and the temperature. The membrane fluidity of the POPC liposome, evaluated by the hydrophobic probe, 1-(4-trimethyl-aminophenyl)-6-diphenyl-1,3,5-hexatriene, was also dependent on the liposome diameter and the temperature. The Ps values for various neutral liposomes under gel phase or liquid-crystalline phase were correlated with their membrane fluidity, although some data were a little scattered, possibly due to the lamellarity. It is therefore considered that the membrane fluidity dominates the permeability of calcein across the neutral phospholipid membrane. Based on the above results, the Ps value for liposomes immobilized on the solid surface is discussed.

Interaction mechanism of mono-PEGylated proteins in electrostatic interaction chromatography.

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

Relationship between the mobility of phosphocholine headgroups of liposomes and the hydrophobicity at the membrane interface: A characterization with spectrophotometric measurements

In this study, we investigated the dynamics of a membrane interface of liposomes prepared by eight zwitterionic phosphatidylcholines in terms of their headgroup mobility, with spectroscopic methods such as dielectric dispersion analysis (DDA), fluorescence spectroscopy. The DDA measurement is based on the response of the permanent dipole moment to a driving electric field and could give the information on the axial rotational Brownian motion of a headgroup with the permanent dipole moment. This motion depended on kinds of phospholipids, the diameter of the liposomes, and the temperature. The activation energy required to overcome the intermolecular force between headgroups of phospholipids depended on the strength of the interaction between headgroups such as hydrogen bonds and/or dipole-dipole interaction. Hydration at the phosphorous group of phospholipid and the molecular order of lipid membrane impaired the interaction between headgroups. Furthermore, the hydrophobicity of membrane surface increased parallel to the increase in headgroup mobility. It is, therefore, concluded that hydration of headgroup promoted its mobility to make the membrane surface hydrophobic. The lipid membrane in liquid crystalline phase or the lipid membrane with the larger curvature was more hydrophobic.

Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity.

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1‐palmitoyl‐2‐oleoyl‐ sn‐glycero‐3‐ phosphocholine). The YADH‐ and NAD+‐containing liposomes (YADH‐NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 °C. The enzyme stability of the YADH‐NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 °C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 °C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 °C than at 45 °C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH‐NADL and YADHL, showed stabilities at both 45 and 50 °C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH‐NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 °C than at 45 °C. This was considered to be because even at 50 °C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.

Binding site and elution behavior of DNA and other large biomolecules in monolithic anion-exchange chromatography

Our previous study has shown that there is a good correlation between the number of charges of DNA (from trimer to 50-mer) and the number of binding sites B in electrostatic interaction chromatography (ion-exchange chromatography, IEC). It was also found that high salt (NaCl) concentration is needed to elute large DNAs (>0.6M). In this paper we further performed experiments with large DNAs (up to 95-mer polyT and polyA) and charged liposome particles of different sizes (ca. 30, 50 and 100 nm) with a monolithic anion-exchange disk in order to understand the binding and elution mechanism of very large charged biomolecules or particles. The peak salt (NaCl) concentration increased with increasing DNA length. However, above 50-mer DNAs the value did not increase significantly with DNA length (ca. 0.65-0.70 M). For liposome particles of different sizes the peak salt concentration (ca. 0.62 M) was similar and slightly lower than that for large DNAs (ca. 0.65-0.70 M). The binding site values (ca. 25-30) are smaller than those for large DNAs. When arginine was used as a mobile phase modulator, the elution position of polyA and polyT became very close whereas in NaCl gradient elution polyT appeared after polyA eluted. This was mainly due to suppression of hydrophobic interaction by arginine.

Theoretical background of monolithic short layer ion-exchange chromatography for separation of charged large biomolecules or bioparticles

The retention and peak spreading in linear gradient elution of charged large biomolecules were investigated by using numerical simulations. Oligo-DNA separation by monolithic anion-exchange chromatography was chosen as a model system. The peak width and the retention were well predicted by using the parameters obtained by gradient elution experiments at different gradient slopes. As the distribution coefficient at the peak retention volume K(R) decreases with increasing molecular size, the peak became sharper for larger DNAs. This is due to very large effective charge (binding site) values of large DNAs (20-60). The peak width was well correlated with K(R) based on the model equation developed for linear gradient elution of proteins. It was shown that the monolithic disk is best suited for very large charged biomolecule separations at high flow velocities with shallow gradients slopes.

PEG chain length impacts yield of solid-phase protein PEGylation and efficiency of PEGylated protein separation by ion-exchange chromatography: Insights of mechanistic models

The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction‐based chromatography (ion‐exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono‐PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387–1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid‐phase PEGylation) and the PEGylated proteins were separated on the same column. Solid‐phase PEGylation inhibited the production of multi‐PEGylated forms and resulted in a relatively low yield of selective mono‐PEGylated form. Pore diffusion may play an important role in solid‐phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.

Drying of Yeasts—Factors Affecting Inactivation During Drying

The inactivation behavior of bakers yeasts during low-temperature hot air drying and freeze drying was investigated. The activity was determined by measuring the fermentative abilities. Inactivation was observed during the falling drying rate period even at low temperature (30°C). As their fermentation activities in suspensions at 30°C are maintained up to a few hours, this decrease was found to be due to dehydration inactivation. Similar dehydration inactivation was also observed during freeze drying. The dehydration inactivation became more significant when the drying rate was increased. The stability of dried yeasts increased with decreasing water content. The addition of sorbitol before drying further improved the stability of dried yeasts.

Aβ/Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane

The amyloid beta protein with 42 amino acid residues (Abeta), which is a causative protein of Alzheimers disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Abeta/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water.

Salt tolerant chromatography provides salt tolerance and a better selectivity for protein monomer separations

Salt tolerant chromatography (STC) is an attractive method as buffer exchange during protein purification processes can be skipped; however, the retention and separation mechanism of such STC are still not fully understood. We carried out linear gradient elution (LGE) experiments of bovine serum albumin (BSA) including its dimer form by using poly‐amine ligand STC. The peak salt concentration IR was measured as a function of normalized gradient slope GH, and the number of binding sites B was determined. The separation performance of monomer and dimer was much higher for STC. The IR values of BSA monomer and dimer for STC were much higher (IR > 0.5M) than those for conventional IEC. The IR values of arginine‐Cl gradient decreased markedly compared to those of NaCl gradient whereas they did not change for conventional IEC. This might be due to combined effects of electrostatic and hydrophobic interaction to the retention of proteins in STC. Adding polyethylene glycol (PEG) into the mobile phase of IEC also increased the retention (salt tolerance) and the resolution of BSA monomer and dimer. Higher viscosity and low solubility of proteins due to PEG were disadvantages of this method. STC with poly‐amine ligand might be also suited for the continuous flow‐through separation of monomer.