Underused Marine Resources: Sudden Properties of Cod Skin Gelatin Gel.

The main object of this work was to characterize the structure and properties of laboratory-made fish gelatin from cod skin in comparison with known commercial gelatins of fish and mammalian origin. This is one way we can contribute to the World Food Program and characterize foodstuff resources from alternative natural sources. Our research was based on the combination of an expanded set of complementary physical-chemical methods to study the similarities and distinctions of hydrogels from traditional and novel gelatin sources from underused marine resources. In this work, we have compared the morphology, supramolecular structure and colloid properties of two commercial (mammalian and fish) gelatins with gelatin we extracted from cold-water cod skin in laboratory conditions. The obtained results are novel, showing that our laboratory-produced fish gelatin is much closer to the mammalian one in terms of such parameters as thermal stability and strength of structural network under temperature alterations. Especially interesting are our experimental observations comparing both fish gelatins: it was shown that the laboratory-extracted cod gelatin is essentially more thermally stable compared to its commercial analogue, being even closer in its rheological properties to the mammalian one.

Ions-Induced Alginate Gelation According to Elemental Analysis and a Combinatorial Approach.

This study considers the potential of elemental analysis of polysaccharide ionotropic gels in elucidating the junction zones for different divalent cations. The developed algorithm ensures the correct separation of contributions from physically adsorbed and structure-forming ionic compounds, with the obtained results scaled to alginate C12 block. Possible versions of chain association into dimers and their subsequent integration into flat junction zones were analyzed within the framework of the "egg-box" model. The application of combinatorial analysis made it possible to derive theoretical relations to find the probability of various types of egg-box cell occurrences for alginate chains with arbitrary monomeric units ratio µ = M/G, which makes it possible to compare experimental data for alginates of different origins. Based on literature data and obtained chemical formulas, the possible correspondence of concrete biopolymer cells to those most preferable for filling by alkaline earth cations was established. The identified features of elemental composition suggest the formation of composite hydrated complexes with the participation of transition metal cations. The possibility of quantitatively assessing ordered secondary structures formed due to the physical sorption of ions and molecules from environment, correlating with the sorption capabilities of Me2+ alginate, was established.

The inhibition of glucose uptake to erythrocytes: microwave dielectric response.

Dielectric spectroscopy has been used in the study and development of non-invasive glucose monitoring (NIGM) sensors, including the range of microwave frequencies. Dielectric relaxation of red blood cell (RBC) cytosolic water in the microwave frequency band has been shown to be sensitive to variations in the glucose concentration of RBC suspensions. It has been hypothesized that this sensitivity stems from the utilization of D-glucose by RBCs. To verify this proposition, RBCs were pretreated with inhibitors of D-glucose uptake (cytochalasin B and forskolin). Then their suspensions were exposed to different D-glucose concentrations as measured by microwave dielectric spectroscopy (MDS) in the 500 MHz-40 GHz frequency band. After incubation of RBCs with either inhibitor, the dielectric response of water in the cytoplasm, and specifically its relaxation time, demonstrated minimal sensitivity to the change of D-glucose concentration in the medium. This result allows us to conclude that the sensitivity of MDS to glucose uptake is associated with variations in the balance of bulk and bound RBC cytosolic water due to intracellular D-glucose metabolism, verifying the correctness of the initial hypothesis. These findings represent a further argument to establish the dielectric response of water as a marker of glucose variation in RBCs.

Hydration of methemoglobin studied by in silico modeling and dielectric spectroscopy.

The hemoglobin concentration of 35 g/dl of human red blood cells is close to the solubility threshold. Using microwave dielectric spectroscopy, we have assessed the amount of water associated with hydration shells of methemoglobin as a function of its concentration in the presence or absence of ions. We estimated water-hemoglobin interactions to interpret the obtained data. Within the concentration range of 5-10 g/dl of methemoglobin, ions play an important role in defining the free-to-bound water ratio competing with hemoglobin to recruit water molecules for the hydration shell. At higher concentrations, hemoglobin is a major contributor to the recruitment of water to its hydration shell. Furthermore, the amount of bound water does not change as the hemoglobin concentration is increased from 15 to 30 g/dl, remaining at the level of ∼20% of the total intracellular water pool. The theoretical evaluation of the ratio of free and bound water for the hemoglobin concentration in the absence of ions corresponds with the experimental results and shows that the methemoglobin molecule binds about 1400 water molecules. These observations suggest that within the concentration range close to the physiological one, hemoglobin molecules are so close to each other that their hydration shells interact. In this case, the orientation of the hemoglobin molecules is most likely not stochastic, but rather supports partial neutralization of positive and negative charges at the protein surface. Furthermore, deformation of the red blood cell shape results in the rearrangement of these structures.

Oxygenation state of hemoglobin defines dynamics of water molecules in its vicinity.

This study focuses on assessing the possible impact of changes in hemoglobin (Hb) oxygenation on the state of water in its hydration shell as it contributes to red blood cell deformability. Microwave Dielectric Spectroscopy (MDS) was used to monitor the changes in interactions between water molecules and Hb, the number of water molecules in the protein hydration shell, and the dynamics of pre-protein water in response to the transition of Hb from the tense (T) to the relaxed (R) state, and vice versa. Measurements were performed for Hb solutions of different concentrations (5 g/dl-30 g/dl) in phosphate-buffered saline buffer. Cole-Cole parameters of the main water relaxation peak in terms of interactions of water molecules (dipole-dipole/ionic dipole) during the oxygenation-deoxygenation cycle were used to analyze the obtained data. The water mobility-represented by α as a function of ln τ-differed dramatically between the R (oxygenated) state and the T (deoxygenated) state of Hb at physiologically relevant concentrations (30 g/dl-35 g/dl or 4.5 mM-5.5 mM). At these concentrations, oxygenated hemoglobin was characterized by substantially lower mobility of water in the hydration shell, measured as an increase in relaxation time, compared to deoxyhemoglobin. This change indicated an increase in red blood cell cytosolic viscosity when cells were oxygenated and a decrease in viscosity upon deoxygenation. Information provided by MDS on the intraerythrocytic water state of intact red blood cells reflects its interaction with all of the cytosolic components, making these measurements powerful predictors of the changes in the rheological properties of red blood cells, regardless of the cause.

Dielectric spectra broadening as a signature for dipole-matrix interactions. V. Water in protein solutions.

In this paper, the fifth of our series focused on the dielectric spectrum symmetrical broadening of water, we consider the solutions of methemoglobin (MetHb) in pure water and in phosphate-buffered saline (PBS). The universal character of the Cole-Cole dielectric response, which reflects the interaction of water dipoles with solute molecules, was described in Paper I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)]. It enables the interpretation of the dielectric data of MetHb solutions in a unified manner using the previously developed 3D trajectory method driven by the protein concentration. It was shown that protein hydration is determined by the interaction of water dipoles with the charges and dipoles located on the rough surfaces of the protein macromolecules. In the case of the buffered solution, the transition from a dipole-charged to a dipole-dipole interaction with the protein concentration is observed {see Paper III [A. Puzenko et al., J. Chem. Phys. 137, 194502 (2012)]}. A new approach is proposed for evaluating the amount of hydration water molecules bounded to the macromolecule that takes into account the number of positive and negative charges on the protein's surface. In the case of the MetHb solution in PBS, the hydration of the solvent ions and their interaction with charges on the protein's surface are also taken into consideration. The difference in hydration between the two solutions of MetHb is discussed.

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots.

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1'E)-12-(1'-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, (1)H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1'E,3'Z)-12-(1',3'-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.

Tomato CYP74C3 is a multifunctional enzyme not only synthesizing allene oxide but also catalyzing its hydrolysis and cyclization.

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)-hydroperoxide with dilute suspensions of LeAOS3 (10-20 s, 0 degrees C) yield mostly the expected allene oxide (12Z)-9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which was detected as its methanol-trapping product. In contrast, the relative yield of 9,10-EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80-fold larger amounts of LeAOS3, while alpha-ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products. Both the alpha-ketol and 10-oxo-PEA were also produced when LeAOS3 was exposed to preformed 9,10-EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)-hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.