Chemical and Physical Characterisation of Macroaggregated Human Serum Albumin: Strength and Specificity of Bonds with 99mTc and 68Ga

Background: Macroaggregated human serum albumin (MAA) properties are widely used in nuclear medicine, labelled with 99mTc. The aim of this study is to improve the knowledge about the morphology, size, dimension and physical–chemical characteristics of MAA and their bond with 99mTc and 68Ga. Methods: Commercial kits of MAA (Pulmocis®) were used. Characterisation through experiments based on SEM, DLS and Stokes’ Law were carried out. In vitro experiments for Langmuir isotherms and pH studies on radiolabelling were performed and the stability of the radiometal complex was verified through competition reactions. Results: The study settles the MAA dimension within the range 43–51 μm. The Langmuir isotherm reveals for [99mTc]MAA: Bmax (46.32), h (2.36); for [68Ga]MAA: Bmax (44.54), h (0.893). Dual labelling reveals that MAA does not discriminate different radioisotopes. Experiments on pH placed the optimal pH for labelling with 99mTc at 6. Conclusion: Radiolabelling of MAA is possible with high efficiency. The nondiscriminatory MAA bonds make this drug suitable for radiolabelling with different radioisotopes or for dual labelling. This finding illustrates the need to continue investigating MAA chemical and physical characteristics to allow for secure labelling with different isotopes.

Development of a combined solution of pyrimidine nucleotides with vitamin B6

Pyrimidine nucleotides, namely uridine monophosphate and cytidine monophosphate, play an important role in the cellular metabolism of nerve fibers. The combination of these nucleotides with pyridoxine hydrochloride (vitamin В6) in one dosage form will allow us to fully implement the strategy of the complex neurotropic pharmacotherapy in neuropathies of various origins. To develop a stable solution, an important step at the stage of the composition development is to study the compatibility of active substances (active pharmaceutical ingredients - APIs) in solution. Samples of binary solutions and solutions containing all active substances were prepared and examined. The API interaction was determined by various parameters – changes in color, transparency, рН, the total impurity content, etc. Based on a comprehensive study of the processes of the API dissolution the optimal pH limits of the solution required for the stable existence of a combination of substances with different pH values of the medium have been substantiated and experimentally confirmed. As a result of the research, the optimal pH value of the solution recommended is 4.0-4.8. In the composition of substances the amount of water can be in an adsorbed or crystallized, or combined form. This fact should be taken into account in order to correctly calculate the actual amounts of initial ingredients when preparing the solution. The nature of the water component of APIs was clarified when studying the phase composition of samples on a powder diffractometer. The studies conducted have allowed us to determine the directions of further research for developing the composition of an injection drug. This research is in choosing the optimal buffer system and excipients-antioxidants.

Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

The optimal pH of AID is skewed from that of its catalytic pocket by DNA-binding residues and surface charge

Activation-induced cytidine deaminase (AID) is a member of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of cytidine deaminases. AID mutates immunoglobulin loci to initiate secondary antibody diversification. The APOBEC3 (A3) sub-branch mutates viral pathogens in the cytosol and acidic endosomal compartments. Accordingly, AID functions optimally near neutral pH, while most A3s are acid-adapted (optimal pH 5.5-6.5). To gain a structural understanding for this pH disparity, we constructed high-resolution maps of AID catalytic activity vs pH. We found AID’s optimal pH was 7.3 but it retained most (>70%) of the activity at pH 8. Probing of ssDNA-binding residues near the catalytic pocket, key for bending ssDNA into the pocket (e.g R25) yielded mutants with altered pH preference, corroborating previous findings that the equivalent residue in APOBEC3G (H216) underlies its acidic pH preference. AID from bony fish exhibited more basic optimal pH (pH 7.5-8.1) and several R25-equivalent mutants altered pH preference. Comparison of pH optima across the AID/APOBEC3 family revealed an inverse correlation between positive surface charge and overall catalysis. The paralogue with the most robust catalytic activity (APOBEC3A) has the lowest surface charge, most acidic pH preference, while the paralogue with the most lethargic catalytic rate (AID) has the most positive surface charge and highest optimal pH. We suggest one possible mechanism is through surface charge dictating an overall optimal pH that is different from the optimal pH of the catalytic pocket microenvironment. These findings illuminate an additional structural mechanism that regulates AID/APOBEC3 mutagenesis.

Biotransformation of Chromium (VI) via a Reductant Activity from the Fungal Strain Purpureocillium lilacinum

Industrial effluents from chromium-based products lead to chromium pollution in the environment. Several technologies have been employed for the removal of chromium (Cr) from the environment, including adsorption, ion-exchange, bioremediation, etc. In this study, we isolated a Cr (VI)-resistant fungus, Purpureocillium lilacinum, from contaminated soil, which could reduce chromium. We also characterized a reductant activity of dichromate found in the cellular fraction of the fungus: optimal pH and temperature, effect of enzymatic inhibitors and enhancers, metal ions, use of electron donors, and initial Cr (VI) and protein concentration. This study also shows possible mechanisms that could be involved in the elimination of this metal. We observed an increase in the reduction of Cr (VI) activity in the presence of NADH followed by that of formate and acetate, as electron donor. This reduction was highly inhibited by EDTA followed by NaN3 and KCN, and this activity showed the highest activity at an optimal pH of 7.0 at 37 °C with a protein concentration of 3.62 µg/mL.

Poly(vinyl alcohol)-based Electrospun Nanofibers: Characterization and Phytase Immobilization

The aim of this study is to immobilization phytase obtained from cowpea seeds into nanofiber-based on poly(vinyl alcohol) (PVA) and to investigate kinetic properties, optimal pH, and temperature of free and immobilized phytase. The structural analysis and morphological properties of the nanofibers are carried out via SEM and XRD. The results indicated that enzyme stability, pH, and thermal stability are increased after immobilizing phytase into the nanofiber. The optimum pH and temperature of the free and immobilized phytase are found as pH 5.0 and 45-65 oC, respectively. These results indicated that the immobilized phytase could be a good candidate for agriculture, animal feed, food, and medical applications.

Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.

Amino-Thiol Bifunctional Polysilsesquioxane/Carbon Nanotubes Magnetic Composites as Adsorbents for Hg(II) Removal

Amino-thiol bifunctional polysilsesquioxane/carbon nanotubes (PSQ/CNTs) magnetic composites were prepared by sol-gel method with two types of functional siloxanes coating on carboxyl CNTs simultaneously. The composites were served as efficient adsorbents for removing Hg(II) in aqueous solution and the adsorption properties were investigated systematically. The optimal pH of bifunctional composites for Hg(II) removal is at pH 4.5. The thermodynamic fitting curves are more consistent with the Langmuir model and the adsorption capacities of the bifunctional composites for Hg(II) varied from 1.63 to 1.94 mmol g−1 at 25°C according to the Langmuir model. The kinetics curves are more fitted to the pseudo-second-order model and the composites could selectively adsorb Hg(II) in a series of binary metal ions solution. The elution regeneration tests showed that the adsorption rate could still reach 78% after repeat cycle three times. It is expected that the bifunctional PSQ/CNTs magnetic composites can be potentially applied to remove low concentration Hg(II) from waste water.