BackgroundRecent research on nanoparticles in a number of crops has evidenced for enhanced germination and seedling growth, physiological activities including photosynthetic activity and nitrogen metabolism, mRNA expression and protein level, and also positive changes in gene expression indicating their potential use in crop improvement. We used a medicinally rich vegetable crop, bitter melon, as a model to evaluate the effects of seed treatment with a carbon-based nanoparticle, fullerol [C60(OH)20], on yield of plant biomass and fruit characters, and phytomedicine contents in fruits.ResultsWe confirmed the uptake, translocation and accumulation of fullerol through bright field imaging and Fourier transform infra-red spectroscopy. We observed varied effects of seed treatment at five concentrations, including non-consequential and positive, on plant biomass yield, fruit yield and its component characters, and content of five phytomedicines in fruits. Fullerol-treatment resulted in increases up to 54% in biomass yield and 24% in water content. Increases of up to 20% in fruit length, 59% in fruit number, and 70% in fruit weight led to an improvement up to 128% in fruit yield. Contents of two anticancer phytomedicines, cucurbitacin-B and lycopene, were enhanced up to 74% and 82%, respectively, and contents of two antidiabetic phytomedicines, charantin and insulin, were augmented up to 20% and 91%, respectively. Non-significant correlation inter se plant biomass, fruit yield, phytomedicine content and water content evidenced for separate genetic control and biosynthetic pathways for production of plant biomass, fruits, and phytomedicines in fruits, and also no impact of increased water uptake.ConclusionsWhile our results indicated possibility of improving crop yield and quality by using proper concentrations of fullerol, extreme caution needs to be exercised given emerging knowledge about accumulation and toxicity of nanoparticles in bodily tissues.
The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s(-1) at 1.5 mL min(-1)). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5%), but increased resolution of ~14% between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s(-1) required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.
Polypropylene (PP) capillary-channeled polymer (C-CP) fiber stationary phases are investigated for applications in HPLC. Specifically, the roles that fiber size and shape, linear velocity, interstitial fraction, and column inner diameter play in separation efficiency were evaluated using a uracil and butylparaben mixture eluted under isocratic conditions. Four fiber types, having nominal diameters ranging from 30 to 65 μm, were used in 250 mm × 2.1 mm columns. Optimum flow characteristics, as judged by plate height and resolution, were observed for 40 μm diameter PP C-CP fibers packed at an interstitial fraction of ~0.63, over a broad range of linear velocities (~2 to 37 mm/s). The influence of column inner diameter was studied on 1.5, 2.1, and 4.6 mm columns packed at the optimal interstitial fraction. The best performing column in terms of plate height and resolution was the 2.1 mm inner diameter. C-CP columns were also evaluated for the separation of a protein mixture composed of ribonuclease A, cytochrome c, and transferrin. Results obtained with the biomacromolecules mixture validate the optimal structural and operative conditions determined with the small solutes, laying the groundwork towards biomacromolecule applications, focusing more on the chemical aspects of separations.
Nylon 6 capillary-channeled polymer (C-CP) fibers are investigated as an alternative support/stationary phase for downstream processing of macromolecules. Ionizable amine and carboxylic acid end groups on the native fiber surface allow for ion exchange chromatography (IEC). The low cost and ability to operate at high linear velocities and low back pressures are practical advantages of C-CP fibers for preparative-scale macromolecule separations. The lack of fiber porosity ensures facile adsorption/desorption that is conducive to high throughput and recoveries/yields. Described here is a preliminary investigation of the processing characteristics of lysozyme on nylon 6 fibers with an eye toward downstream processing applications. Fibers were packed into microbore (0.8 mm i.d.) and analytical-size (2.1 mm i.d.) columns for the evaluation of the role of linear velocity on pressure drop, frontal throughput, and yield. Protein isolation by frontal development involved three steps: loading of the column to breakthrough, an aqueous wash, and a salt wash to recover the protein. Frontal throughput was evaluated with different salt concentrations (0-1000 mM NaCl) and different linear velocities (6-24 mm s(-1)). The observed throughput values are in the range of 0.12-0.20 mg min(-1) when 0.25 mg mL(-1) lysozyme (in 20 mM Tris-HCl) is loaded onto 78 mg of C-CP fiber in 0.52 mL volume analytical columns. Increased throughput and yield were found when protein was loaded and eluted at high linear velocity. Results of this study lend credence to the further development of C-CP fibers for biomacromolecule processing on larger scales.
The analysis of metal-binding proteins requires careful sample manipulation to ensure that the metal-protein complex remains in its native state and the metal retention is preserved during sample preparation or analysis. Chemical analysis for the metal content in proteins typically involves some type of liquid chromatography/electrophoresis separation step coupled with an atomic (i.e., inductively coupled plasma-optical emission spectroscopy or -mass spectrometry) or molecular (i.e., electrospray ionization-mass spectrometry) analysis step that requires altered-solvent introduction techniques. UV-VIS absorbance is employed here to monitor the iron content in human holo-transferrin (Tf) under various solvent conditions, changing polarity, pH, ionic strength, and the ionic and hydrophobic environment of the protein. Iron loading percentages (i.e. 100% loading equates to 2 Fe(3+):1 Tf) were quantitatively determined to evaluate the effect of solvent composition on the retention of Fe(3+) in Tf. Maximum retention of Fe(3+) was found in buffered (20 mM Tris) solutions (96 ± 1%). Exposure to organic solvents and deionized H(2)O caused release of ~23-36% of the Fe(3+) from the binding pocket(s) at physiological pH (7.4). Salt concentrations similar to separation conditions used for ion exchange had little to no effect on Fe(3+) retention in holo-Tf. Unsurprisingly, changes in ionic strength caused by additions of guanidine HCl (0-10 M) to holo-Tf resulted in unfolding of the protein and loss of Fe(3+) from Tf; however, denaturing and metal loss was found not to be an instantaneous process for additions of 1-5 M guanidinium to Tf. In contrast, complete denaturing and loss of Fe(3+) was instantaneous with ≥6 M additions of guanidinium, and denaturing and loss of iron from Tf occurred in parallel proportions. Changes to the hydrophobicity of Tf (via addition of 0-14 M urea) had less effect on denaturing and release of Fe(3+) from the Tf binding pocket compared to changes in ionic strength.
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