Self-assembled nanocages for hydrophilic guest molecules

Reverse polymeric micelles are obtained following the association of polymeric amphiphiles in apolar media. To this date, reports of pharmaceutical applications for such micelles have been scarce, mainly because these systems have been studied in solvents that are not suitable for medical use. Here, alkylated star-shaped poly(glycerol methacrylate) polymers have been proposed in the design of oil-soluble reverse polymeric micelles. Micellar behavior was studied in various apolar solvents, including ethyl oleate, a pharmaceutically acceptable vehicle. The polymers were shown to assemble into spherical nanostructures (<40 nm) as determined by cryogenic transmission electron microscopy and atomic force microscopy studies. Interestingly, the reverse micelles were able to encapsulate various peptides/proteins (vasopressin, myoglobin, and albumin) in substantial amounts and facilitate their solubilization in oil. The nature of both the polymer used in micelle formation and the guest molecules was found to influence the ability of the micelle to interact with hydrophilic compounds.     

Unusual peroxidase activity of a myoglobin mutant with two distal histidines

By retaining the native distal His64 in sperm whale myoglobin(Mb),a second distal histidine was engineered in Mb by mutating Leu29 to His29.The resultant mutant of L29H Mb exhibits an unusual enhanced peroxidase activity with a positive cooperativity in comparison to that of wild type Mb.The new enzyme with two cooperative distal histidines has not been found in native peroxidase, which emphasizes a creation of the rational protein design.     

Direct comparison of electron transfer properties of two distinct semisynthetic triads with non-protein based triad: unambiguous experimental evidences on protein matrix effects

In order to understand the roles of protein matrix in electron transfer processes (ET) within biological systems, a heme-based donor (Zn-heme: ZnPP)-sensitizer (Ru2+(bpy)3)-acceptor (cyclic viologen: BXV4+) triad 1 was used as a probe molecule. Two semi-synthetic systems, Cyt-b562(1) and Mb(1), in which the triad is incorporated into cytochrome b562 (Cyt-b562) or into myoglobin (Mb), were constructed by cofactor reconstitution. These two semi-synthetic proteins were compared with the triad itself (i.e., without the protein matrix) using absorption spectroscopy, steady state emission and excitation studies, laser flash photolysis experiments, and molecular modeling. Photoexcitation of the ZnPP moiety of Cyt-b562(1) or Mb(1) leads to a direct ET from the triplet state of ZnPP state (3ZnPP) to BXV4+ to generate a final charge-separated (CS) state, Cyt-b562(Zn+)-Ru2+-BXV3+* or Mb(Zn+)-Ru2+-BXV3+*. On the other hand, direct ET from the excited ZnPP moiety to the BXV4+ moiety is also involved in 1 in the absence of the protein matrix, but the excited state of ZnPP involved is not 3ZnPP, but the singlet excited state (1ZnPP) in this pathway. When the Ru2+(bpy)3 moiety of Cyt-b562(1) or Mb(1) is excited, a stepwise ET relay occurs with the ion-pair, Cyt-b562(Zn)-Ru3+-BXV3+* or Mb(Zn)-Ru3*-BXV3+*, as an intermediate, leading to the same final CS state as that generated in the direct ET pathway. The lifetimes of the corresponding final CS states were determined to be 300 ns for 1 in the absence of the protein matrix, 600-900 ns for Cyt-b562(1) and 1.1-18 micros for Mb(1), the values of which are greatly affected by the protein matrix. Molecular modeling study of the three systems consistently explained the differences of their photophysical behavior.     

Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers

Films constructed layer-by-layer on electrodes with architecture {protein/hyaluronic acid (HA)}n containing myoglobin (Mb) or horseradish peroxidase (HRP) were protected against protein damage by H2O2 by using outer catalase layers. Peroxidase activity for substrate oxidation requires activation by H2O2, but {protein/HA}n films without outer catalase layers are damaged slowly and irreversibly by H2O2. The rate and extent of damage were decreased dramatically by adding outer catalase layers to decompose H2O2. Comparative studies suggest that protection results from catalase decomposing a fraction of the H2O2 as it enters the film, rather than by an in-film diffusion barrier. The outer catalase layers controlled the rate of H2O2 entry into inner regions of the film, and they biased the system to favor electrocatalytic peroxide reduction over enzyme damage. Catalase-protected {protein/HA}n films had an increased linear concentration range for H2O2 detection. This approach offers an effective way to protect biosensors from damage by H2O2.     

Self-assembled architectures from biohybrid triblock copolymers

The synthesis and self-assembly behavior of biohybrid ABC triblock copolymers consisting of a synthetic diblock, polystyrene-b-polyethylene glycol (PSm-b-PEG113), where m is varied, and a hemeprotein, myoglobin (Mb) or horse radish peroxidase (HRP), is described. The synthetic diblock copolymer is first functionalized with the heme cofactor and subsequently reconstituted with the apoprotein or the apoenzyme to yield the protein-containing ABC triblock copolymer. The obtained amphiphilic block copolymers self-assemble in aqueous solution into a large variety of aggregate structures. Depending on the protein and the polystyrene block length, micellar rods, vesicles, toroids, figure eight structures, octopus structures, and spheres with a lamellar surface are formed.     

Thermostable biocatalytic films of enzymes and polylysine on electrodes and nanoparticles in microemulsions

Microemulsions of oil, water and surfactant were evaluated as media for biocatalysis at high temperatures employing films of polylysine (PLL) and the enzymes horseradish peroxidase (HRP), soybean peroxidase (SBP) and the protein myoglobin (Mb). PLL was covalently linked to oxidized pyrolytic graphite electrodes or carboxylated 500 nm diameter silica nanoparticles, then cross-linked by amidization to HRP, SBP and Mb. The resulting film systems were stable at 90 degrees C for >12 h in microemulsions. Characterization of the microemulsions by conductivity, viscosity and probe diffusion coefficients suggested that these media have bicontinuous microstructures from 25 to 90 degrees C. UV circular dichroism and visible spectroscopy confirmed that the enzymes retained near-native conformation in the films at temperatures as high as 90 degrees C. Oxidation of o-methoxyphenol to 3,3'-dimethoxy-4,4'-biphenoquinone by enzyme-PLL films on silica nanoparticles gave yields 3-5-fold larger in microemulsions at 90 degrees C compared to the same reaction at 25 degrees C. The best yields were in CTAB microemulsions and were 3-fold larger than in buffers at 90 degrees C.     

Oxygen‐binding Protein Fiber and Microgel: Supramolecular Myoglobin–Poly(acrylate) Conjugates

A supramolecular conjugate of myoglobin (Mb) and water‐soluble poly(acrylate), (PA_5k and PA_25k, where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme‐heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme‐propionates is coupled to the side‐chain carboxyl groups of poly(acrylate) activated by N‐hydroxysuccinimide. The ratios of the heme‐modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme‐PA_5k and Heme‐PA_25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb‐connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme‐modified polymer and dimeric apomyoglobin as a cross‐linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.     

The direct electron transfer of myoglobin based on the electron tunneling in proteins

The electron tunneling of the protein-polypeptide interactions was observed in the study of direct electron transfer of the myoglobin (Mb) on the electrode surface. The Mb was selected as a redox active protein and gelatine was selected to couple with Mb to form an electron tunneling. The electrochemical results indicated the presence of the electron tunneling and the direct electron transfer. The circular dichroism spectra suggested that the beta-sheet chain of gelatine could interact with alpha-helical chain to form an electron tunneling to promote the protein direct electrochemistry. The SDS-PAGE results proved that the electron tunneling between Mb and gelatine was noncovalent hydrogen bonds. The immobilized Mb showed a couple of quasi-reversible redox peaks with a formal potential of -0.37V (vs SCE) in 0.1 M pH 7.0 PBS. The modified electrodes displayed a rapid amperometric response to the reduction of oxygen, H2O2, and nitrite.     

Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.     

Immobilization of peroxidase on SiO2 surfaces with the help of a dendronized polymer and the avidin-biotin system

Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications.     

Macromolecule-to-Amphiphile Conversion Process of a Polyoxometalate–Polymer Hybrid and Assembled Hybrid Vesicles

We report our findings on the macromolecule-to-amphiphile conversion process of a polyoxometalate–polymer hybrid and the assembled hybrid vesicles formed by aggregation of the hybrid amphiphile. The polyoxometalate–polymer hybrid is composed of a polyoxometalate (POM) cluster, which is covered by five tetrabutylammonium (Bu₄N⁺) countercations, and a polystyrene (PS) chain. Through a cation-exchange process the Bu₄N⁺ countercations can be replaced by protons to form a hybrid amphiphile composed of a hydrophilic, protonated POM cluster and a hydrophobic PS chain. By implementing a directed one-dimensional diffusion and analyzing the diffusion data, we confirmed that the diffusion of solvated protons rather than macromolecules or aggregates is the key factor controlling the conversion process. Once the giant hybrid amphiphiles were formed, they immediately assembled into kinetically favored vesicular aggregates. During subsequent annealing these vesicular aggregates were transformed into thermodynamically stable vesicular aggregates with a perfect vesicle structure. The success in the preparation of the POM-containing hybrid vesicles provides us with an opportunity of preparing POM-functionalized vesicles.     

Fabrication and biocompatible characterization of magnetic hollow capsules

Monodispersed Fe3O4/polypyrrole （PPy） hollow particles were synthesized via controllable in-situ deposition and polymerization techniques using poly（styrene-co-acrylic） （PSA） latex as template. Field-dependent magnetization plot illustrates that the capsules are superparamagnetic at 300 K. FTIR spectrum confirms that the myoglobin （Mb） molecule adsorbed on the surface of Fe3O4/PPy hollow particle essentially retains its native structure. Furthermore, direct electrochemistry of Mb can be realized on Fe3O4/PPy capsules modified pyrolytic graphite disk electrode, which indicates that the magnetic conductive polymer capsules can promote the electron transfer of protein.     

Electrochemical studies on reconstituted horseradish peroxidase modified carbon paste electrodes

Horseradish peroxidase (HRP) is a heme protein that acts specifically on H(2)O(2) as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Teale's method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H(2)O(2) using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 microM H(2)O(2). The rec-HRP paste showed approximately 100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 microM was observed.     

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4 °C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20–30 µm as well as below 30 nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.     

Enzyme‐Initiated Free‐Radical Polymerization of Molecularly Imprinted Polymer Nanogels on a Solid Phase with an Immobilized Radical Source

An enzyme‐mediated synthetic approach is described for the preparation of molecularly imprinted polymer nanoparticles (MIP‐NPs) in aqueous media. Horseradish peroxidase (HRP) was used to initiate the polymerization of methacrylate or vinyl monomers and cross‐linkers by catalyzing the generation of free radicals. To prevent entrapment of the enzyme in the cross‐linked polymer, and to enable it to be reused, HRP was immobilized on a solid support. MIPs based on 4‐vinylpyridine and 1,4‐bis(acryloyl)piperazine for the recognition of 2,4‐dichlorophenoxyacetic acid (2,4‐D) and salicylic acid were synthesized in an aqueous medium. MIPs for the protein trypsin were also synthesized. MIP nanoparticles with sizes between 50 and 300 nm were obtained with good binding properties, a good imprinting effect, and high selectivity for the target molecule. The reusability of immobilized HRP for MIP synthesis was shown for several batches.     

Coherence spectroscopy investigations of the low-frequency vibrations of heme: effects of protein-specific perturbations

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above approximately 50 cm(-1) and very weak activity below approximately 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T approximately < 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.     

Insight into Protein–Polymer Conjugate Relaxation Dynamics: The Importance of Polymer Grafting

The bio and chemical physics of protein–polymer conjugates are related to parameters that characterize each component. With this work, it is intended to feature the dynamical properties of the protein–polymer conjugate myoglobin (Mb)–poly(ethyl ethylene phosphate), in the ps and ns time scales, in order to understand the respective roles of the protein and of the polymer size in the dynamics of the conjugate. Elastic and quasi‐elastic neutron scattering is performed on completely hydrogenated samples with variable number of polymer chains covalently attached to the protein. The role of the polymer length in the protein solvation and internal dynamics is investigated using two conjugates formed by polymers of different molecular weight. It is confirmed that the flexibility of the complex increases with the number of grafted polymer chains and that a sharp dynamical transition appears when either grafting density or polymer molecular weight are high. It is shown that protein size is crucial for the polymer structural organization and interaction on the protein surface and it is established that the glass properties of the polymer change upon conjugation. The results give a better insight of the equivalence of the polymer coating and the role of water on the surface of proteins.     

Anisotropic orientation of horseradish peroxidase by reconstitution on a thiol-modified gold electrode

Horseradish peroxidase (HRP) was reconstituted on the surface of a gold electrode that was modified first with a hemin-carbon-chain-thiol derivative followed by addition of the apo protein to the contacting solution. To facilitate the reconstitution of the holo enzyme, the hemin needs to be immobilised on a carbon-chain spacer arm. To achieve this, an immobilisation protocol was developed that is based on the initial formation of a mixed self-assembled monolayer on the gold surface consisting of 3-carboxypropyl disulphide and an activated disulphide (3,3'-dithiodipropionic acid di-(N-succinimidyl ester)) followed by binding of a diaminoalkane to the activated disulphide. The hemin was then coupled to the second amino group of the diaminoalkane by means of a carbodiimide coupling reagent. Finally, the enzyme was reconstituted on the hemin-modified surface by immersion of the electrode in a solution containing apo-HRP. The advantage of this method is that the length of the spacer arm can be changed easily, because diaminoalkanes of different chain lengths are available. The electrochemistry of the hemin and the reconstituted HRP electrodes was studied by means of cyclic voltammetry and differential-pulse voltammetry. The catalytic ability for reduction of hydrogen peroxide was investigated for both direct and mediated electrochemistry with a soluble electron donor (ortho-phenylenediamine).     

Hybridization of modified-heme reconstitution and distal histidine mutation to functionalize sperm whale myoglobin

To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D.2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D.2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.     

Photochemical activation of a polycarbonate surface for covalent immobilization of a protein ligand

Polycarbonate—a thermostable polymer is activated by a simple and rapid method using a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) for covalent immobilization of a biomolecule. Horseradish peroxidase (HRP) is used as a model enzyme to check the efficacy of the activated surface. HRP is immobilized on the activated polycarbonate surface without addition of any reagent or catalyst and is found to give 2-2.5-fold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. Photochemical attachment of FNAB to the PC surface is confirmed by X-ray photoelectron spectroscopy (XPS), which shows the presence of nitrogen and fluorine in the ratio of 2:1 in the activated polycarbonate. Disappearance of fluorine peak in the XP spectra of PC bound enzyme further confirms the covalent binding of HRP, through displacement of fluorine moiety of the activated PC by the amino group of the protein. Optimized concentration of the photolinker is found as 6 μmol of FNAB per well and time of photo irradiation is 8 min for activation of a PCR polycarbonate plate. PC bound HRP has shown enhanced thermal and storage stability. Kinetic studies of the immobilized HRP shows improved catalytic activity. The potential application of activated polycarbonate surface includes immobilization of biomolecules for biosensors, immunoassays, and protein and DNA micro-arrays. Due to the stability of the polycarbonate at high temperature, the activated polycarbonate has an advantage for immobilization of thermostable biomolecule such as thermostable enzyme for reaction at elevated temperature.