Stimuli-responsive separation of proteins using immobilized liposome chromatography.

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s),max = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s),max = 11.3 at pH 4; carbonic anhydrase from bovine, k(s),max = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the ks-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.

Immobilized liposome chromatography for refolding and purification of protein.

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield.

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes.

Heating of several protein solutions at 40-47 degrees C for 5-60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of beta-gal at various temperatures were maximized under suitable heating conditions (45 degrees C, 30 min). Those of alpha-glu and CAB were maximized at 40-45 and 60 degrees C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of beta-gal, alpha-glu and CAB.

Model system for heat-induced translocation of cytoplasmic beta-galactosidase across phospholipid bilayer membrane.

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.

The Protein Data Bank. A computer-based archival file for macromolecular structures.

The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.

Interpretation of the doublet at 850 and 830 cm-1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds.

The doublet at 850 and 830 cm-1 in the Raman spectra of proteins containing tyrosyl residues has been examined as to its origin and the relation of its components to the environment of the phenyl ring, the state of the phenolic hydroxyl group, and the conformation of the amino acid backbone. Raman spectral studies on numerous model molecules related to tyrosine, including certain deuterium derivatives, show that the doublet is due to Fermi resonance between the ring-breathing vibration and the overtone of an out-of-plane ring-bending vibration of the para-substituted benzenes. Further examination of the effects of pH and solvents on the Fermi doublet and of the crystallographic data demonstrates that the intensity ratio of the two components depends on changes in the relative frequencies of the two vibrations. These in turn are found to be sensitive to the nature of the hydrogen bonding of the phenolic hydroxyl group of its ionization, but much less so to the environment of the phenyl ring and the conformation of the amino acid backbone. By use of the relative intensities of the doublet in model systems where the phenolic hydroxyl group is strongly hydrogen-bonded, weakly hydrogen-bonded, free or ionized, the reported Raman intensities of the doublets observed in the Raman spectra of several proteins have been interpreted. The results are compared with those obtained by other techniques.