Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.

Lithium-sulfur (Li-S) batteries are faced with practical drawbacks of poor cycle life and low charge efficiency which hinder their advancements. Those drawbacks are primarily caused by the intrinsic issues of the cathodes (sulfur) and the anodes (Li metal). In attempt to resolve the issues found on the cathodes, this work discusses the method to prepare a binder-free three-dimensional carbon nanotubes-sulfur (3D CNTs-S) composite cathode by a facile and a scalable approach. Here, the 3D structure of CNTs serves as a conducting network to accommodate high loading amounts of active sulfur material. The efficient electron pathway and the short Li ions (Li+) diffusion length provided by the 3D CNTs offset the insulating properties of sulfur. As a result, high areal and specific capacities of 8.8 mAh cm−2 and 1068 mAh g−1, respectively, with the sulfur loading of 8.33 mg cm−2 are demonstrated; furthermore, the cells operated at a current density of 1.4 mA cm−2 (0.1 C) for up to 150 cycles. To address the issues existing on the anode part of Li-S batteries, this work also covers the novel approach to protect a Li metal anode with a thin layer of two-dimensional molybdenum disulfide (MoS2). With the protective layer of MoS2 preventing the growth of Li dendrites, stable Li electrodeposition is realized at the current density of 10 mA cm−2; also, the MoS2 protected anode demonstrates over 300% longer cycle life than the unprotected counterpart. Moreover, the MoS2 layer prevents polysulfides from corroding the anode while facilitating a reversible utilization of active materials without decomposing the electrolyte. Therefore, the MoS2 protected anode enables a stable cycle life of over 500 cycles at 0.5 C with the high sulfur loading amount of ~7 mg cm−2 (~67 wt% S content in cathode) under the low electrolyte/sulfur (E/S) ratio of 6 μL mg−1. This translates to the specific energy and power densities of ~550 Wh kg-1 and ~300 W kg−1, respectively. Additionally, such values far exceed the electrochemical performance of the current Li-ion batteries. Therefore, the synergetic effect of utilizing the 3D CNT-S cathode and the MoS2 protected Li anode will allow the Li-S batteries to become applicable for the transportation and the large-scale energy grid applications.

Protamine, a set of small basic proteins (P1 and P2), play a key role in compacting and protecting the DNA in sperm. As such, the structure of how P1 and P2 bind to DNA and potentially themselves and each other is of interest to several fields including forensics. In forensic DNA analysis, protamine binding of DNA is taken advantage of in the “differential extraction” procedure in which a sample that contains sperm and non-sperm cells can have DNA from the two different cell types separated and extracted at different points thus preventing a mixture of DNA. A key component of this greater structure and what makes the differential extraction functional are the disulfide bonds formed by protamine. So as a first step to elucidating the protamine-DNA complex, methods to analyze human protamine via 2D-Liquid Chromatography-Mass Spectrometry (2D-LC-MS) were developed in the hopes they could be used for disulfide bond mapping. Methods and multiple strategies for digestion, 2D-LC-MS were investigated and developed using chum salmon protamine. Digestion strategies were developed for Chymotrypsin and Lys-C, Trypsin or Arg-C with incubation times and substrate:enzyme mass ratios optimized. Various “trap and elute” 2D-chromatography configuration were tested for analysis intact and digested protein. Using H2O with 2% NH4OH as the loading mobile phase and H2O and Acetonitrile both with 0.5% formic acid as the eluting mobile phases with the first dimension column being an HLB (Hydrophilic Lipophilic Balance) 2.1 x 30 mm column and the second dimension being an C18 2.1 x 100 mm was found to produce the highest signal.

The oxidation of thiols in petroleum is a subject of ongoing research, discussion and removal of sulfur is a topic of ongoing legislation. The Merox® process requires high pressures and temperatures. Novel catalysts and methods innovations are of interest. This work examines the synthesis, purification and use of sodium cobalt(II) tetrasulfophthalocyanine to oxidize ethanethiol to diethyl disulfide. Many systems using phthalocyanines carry out the oxidation reaction under basic conditions. This work oxidized ethanethiol to diethyl disulfide in dimethylformamide using cobalt tetrasulfophthalocyanine (CoTSPc) under alkali free conditions and was compared to cobalt sulfate heptahydrate, cobalt phthalocyanine (CoPc), FeTSPc and CuTSPc. The reaction was carried out in oxygen saturated DMF while stirring at 15, 25 and 40.00 °C. The amount of ethanethiol remaining over time was determined using Ellman's reagent. A simple GC method quantified the amount of diethyl disulfide. The reaction proceeded to completion within 10 min at 40.00 °C. A turn over number of 72 and frequency of 8.1 min−1 is obtained. The activation energy was approximately 32 kJ/mol. The prepared CoTSPc catalyst was most catalytic toward oxidation of ethanethiol followed by cobalt sulfate heptahydrate, CoPc, FeTSPc and CuTSPc was non-catalytic.

No description available.

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Engineering. Johannesburg, 21 July 2016 / The use of supercapacitors for energy storage is an attractive approach considering their ability to deliver high levels of electrical power, unlimited charge/discharge cycles, green environmental protection and long operating lifetimes. Despite the satisfactory power density, supercapacitors are yet to match the energy densities of batteries and fuel cells, reducing the competitiveness as a revolutionary energy storage device. Therefore, the biggest challenge for supercapacitors is the trade-off between energy density and power density. This presents an opportunity to enhance the electrochemical capacitance and mechanical stability of an electrode. Previous attempts to get around the problem include developing porous nanostructured electrodes with extremely large effective areas. One of the emerging high-power supercapacitor electrode materials is molybdenum disulfide (MoS2), a member of the transition-metal dichalcogenides (TMDs). Its higher intrinsic fast ionic conductivity and higher theoretical capacity have attracted a lot of attention, particularly in supercapacitors. In addition to double-layer capacitance, diffusion of the ions into the MoS2 at slow scan rates gives rise to Faradaic capacitance. Analogous to Ru in RuO2, the Mo center atom displays a range of oxidation states from +2 to +6. This plays an important role in enhancing charge storage capabilities. However, the electronic conductivity of MoS2 is still lower compared to graphite, and the specific capacitance of MoS2 is still very limited when used alone for energy storage applications. As evident in several literature reports, there is a need to improve the capacitance of MoS2 with conductive materials such as carbon nanotubes (CNT), polyaniline (PANI), polypyrrole (PPy), and reduced graphene (r-GO). Carbon nanospheres (CNS) have, in the past, improved the conductivity of cathode material in Li-ion batteries, owing to their appealing electrical properties, chemical stability and high surface area. The main objective of this dissertation research is to develop nanocomposite materials based on molybdenum sulphide with carbon nanospheres for pseudocapacitors with simultaneously high power density and energy density at low production cost. The research was carried out in two phases, namely, (i) Symmetric pseudocapacitors based on molybdenum disulfide (MoS2)-modified carbon nanospheres: Correlating physico-chemistry and synergistic interaction on energy storage and (ii) The effects of morphology re-arrangements on the pseudocapacitive properties of mesoporous molybdenum disulfide (MoS2) nanoflakes. The physico-chemical properties of the MoS2 layered materials have been interrogated using the surface area analysis (BET), scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), Raman, fourier-transform infrared (FTIR) spectroscopy, and advanced electrochemistry including cyclic voltammetry (CV), galvanostatic cycling with potential limitation (GCPL), repetitive electrochemical cycling tests, and electrochemical impedance spectroscopy (EIS). In the first phase, Molybdenum disulfide-modified carbon nanospheres (MoS2/CNS) with two different morphologies (spherical and flower-like) have been synthesized using hydrothermal techniques and investigated as symmetric pseudocapacitors in aqueous electrolyte. The two different MoS2/CNS layered materials exhibit unique differences in morphology, surface areas, and structural parameters, which have been correlated with their electrochemical capacitive properties. The flower-like morphology (f-MoS2/CNS) shows lattice expansion (XRD), large surface area (BET analysis), and small-sized nanostructures (corroborated by the larger FWHM of the Raman and XRD data). As a contrast to the f-MoS2/CNS, the spherical morphology (s-MoS2/CNS) shows lattice contraction, small surface area with relatively large-sized nanostructures. The presence of CNS on the MoS2 structure leads to slight softening of the characteristic Raman bands (E12g and A1g modes) with larger FWHM. The MoS2 and its CNS-based composites have been tested in symmetric electrochemical capacitors in aqueous 1 M Na2SO4 solution. CNS improves the conductivity of the MoS2 and synergistically enhanced the electrochemical capacitive properties of the materials, especially the f-MoS2/CNS-based symmetric cells (most notably, in terms of capacitance retention). The maximum specific capacitance for f-MoS2/CNS-based pseudocapacitor show a maximum capacitance of 231 F g-1 with high energy density 26 Wh kg-1 and power density 6443 W kg-1. For the s-MoS2/CNS-based pseudocapcitor, the equivalent values are 108 F g-1, 7.4 Wh kg-1 and 3700 W kg-1. The high-performance of the f-MoS2/CNS is consistent with its physico-chemical properties as determined by the spectroscopic and microscopic data. In the second phase, Mesoporous molybdenum disulfide (MoS2) with different morphologies has been prepared via a hydrothermal method using different solvents, water or water/acetone mixtures. The MoS2 obtained with water alone gave graphene-like nanoflakes (g-MoS2) while the other with water/acetone (1:1 ratio) gave a hollow-like morphology (h-MoS2). Both materials are modified with carbon nanospheres as conductive materials and investigated as symmetric pseudocapacitors in aqueous electrolyte (1 M Na2SO4 solution). Interestingly, a simple change of synthesis solvents confers on the MoS2 materials different morphologies, surface areas, and structural parameters, correlated by electrochemical capacitive properties. The g-MoS2 exhibits higher surface area, higher capacitance parameters (specific capacitance of 183 F g-1, maximum energy density of 9.2 Wh kg-1 and power density of 2.9 kW kg-1) but less stable electrochemical cycling compared to the h-MoS2. These findings have opened doors for further exploration of the synergistic effects between MoS2 graphene-like sheets and CNS for energy storage. / MT2017

Protein engineering has been used to alter the stability of proteins for several decades withmuch success, one approach being to introduce two cysteine residues that together form adisulfide bridge. The disulfide bridge can increase the Gibbs free energy of the transitionstate, thus increasing energy difference between the folded state and the unfolding transitionstate, leading to increased kinetic stability of the protein. Subtilisin Carlsberg is a serineprotease that has widespread applications within the industry but has also been tried in biogasprocesses to increase the biomethane yield from proteinaceous substrates. Subtilisin’s activitylifetime was found to be short in the biogas process, which prompted the need to increase theenzyme’s kinetic stability, meaning that the introduction of a disulfide bridge could be asolution. The aim of this project was to increase the kinetic stability of Subtilisin Carlsbergwith the use of introduced disulfide bridges.The production of Subtilisin Carlsberg has traditionally been done using the source organismBacillus Licheniformis, but here a successful method for expressing Subtilisin, and fourdisulfide variants of it, as an inclusion-body protein is presented. Also, a method forpurifying and refolding the protein under denaturing conditions is presented with a significantprotein yield.Thermal stability analysis of the WT enzyme and its four variants (A24C/S86C,N122C/A227C, K12C/E270C, V26C/A231C) was performed using NanoDSF, and showedthat the thermal stability was practically unchanged for A24C/S86C at 67.9 ℃, decreased by5.6 ℃ for N122C/A227C, increased by 8.2 ℃ for K12C/E270C, and increased by 11.5 ℃ forV26C/A231C.The kinetic stability of Subtilisin and its variants was analysed using stopped-flowmeasurements of the proteins’ denaturation rate at various GuHCl concentrations. The resultsshowed that N122C/A227C and V26C/A231C were more kinetically stable than the WTenzyme, while A24C/S86C and K12C/E270C were less stable. N122C/A227C had anactivation energy for unfolding of 5.217 kJ/mol higher than WT Subtilisin. V26C/A231C hadan activation energy for unfolding of 1.220 kJ/mol higher than WT Subtilisin. The resultsthereby show that two disulfides bond mutations achieved the desired outcome of increasedkinetic stability. Thereby, the aim of the project was fulfilled.

A global analysis of the human proteome demonstrates that there are ~5500 tryptic fragments that contain four cysteines in close proximity. Elucidating whether they form disulfide bonds in vivo under different conditions is particularly important because cysteines are known to be a vital cellular redox sensor as well as a catalytic site for important biochemical reactions. However, currently there are no methods that can resolve disulfide patterns in closely-packed cysteine residues from a complex sample. In order to address this problem, we have developed a novel mass-spectrometry-based method to identify the different disulfide bonding patterns possible, using SNAP25B cysteine-rich region as a test case. Unlike traditional proteomics, this method uses non-reduced sample preparation, thus preserving intact disulfide bonds. It relies on collision-induced dissociation (CID) to cause double-backbone and heterolytic disulfide-bond cleavage and compares this to the theoretical MS/MS spectra. CID in an ion trap gives robust detection of double backbone cleavages and heterolytic disulfide-bond cleavages. Here, we report, for the first time, identification of all three disulfide patterns for double-disulfide species of SNAP25B using collision-induced dissociation.

The intensity dependent refractive index change of a medium is frequently described in terms of the product n₂ · I where n₂ is the nonlinear refractive index and I the light intensity. The nonlinear refractive index is often treated as constant which is a reasonable assumption if the light interacts only with bound electrons. In the case of carbon disulfide (CS₂) however, nuclear motions contribute to n₂. These motions occur on the sub picosecond time scale and thus become especially relevant for ultrashort laser pulses. The neat liquid CS₂ is studied because it exhibits a large nonlinear refractive index in comparison to other liquids. Therefore, it is employed in optical switching, optical limiting, and beam filamentation applications. This thesis presents effective n₂ values for Gaussian shaped linearly polarized pulses with central wavelength at [lambda]= 700nm. A theoretical model describing the time evolution of the material response is applied to distinguish between the instantaneous electronic, the ultrafast nuclear and the slow nuclear origins of the nonlinear refractive index. Moreover, the tensor nature of the material response function is studied by means of circularly polarized light. The relative magnitudes of bound electronic and nuclear contributions to n₂ are experimentally determined. Eventually, the dispersion of the instantaneous electronic response is measured in the spectral range between 390nm and 1064nm.

Novel two dimensional nanoscale materials like graphene and metal dichalcogenides (MX2) have attracted the attention of the scientific community, due to their rich physics and wide range of potential applications. It has been shown that novel graphene based transparent conductors and radiofrequency transistors are competitive with the existing technologies. Graphene’s properties are influenced sensitively by adsorbates and substrates. As such not surprisingly, physical properties of graphene are found to have a large variability, which cannot be controlled at the synthesis level, reducing the utility of graphene. As a part of my doctorate dissertation, I have developed atomic hydrogen as a novel technique to count the scatterers responsible for limiting the carrier mobility of graphene field effect transistors on silicon oxide (SiO2) and identified that charged impurities to be the most dominant scatterer. This result enables systematic reduction of the detrimental variability in device performance of graphene. Such sensitivity to substrates also gives an opportunity for engineering device properties of graphene using substrate interaction and atomic scale vacancies. Stacking graphene on hexagonal boron-nitride (h-BN) gives rise to nanoscale periodic potential, which influences its electronic graphene. Using state-of-the-art atomic-resolution scanning probe microscope, I correlated the observed transport properties to the substrate induced extrinsic potentials. Finally in efforts to exploit graphene’s sensitivity to discover new sensor technologies, I have explored noncovalent functionalization of graphene using peptides. Molybdenum disulfide (MoS2) exhibits thickness dependent bandgap. Transistors fabricated from single layer MoS2 have shown a high on/off ratio. It is expected that ad-atom engineering can be used to induce on demand a metal-semiconductor transition in MoS2. In this direction, I have iii explored controlled/reversible fluorination and hydrogenation of monolayer MoS2 to potentially derive a full range of integrated circuit technology. The in-depth characterization of the samples is carried out by Raman/photoluminescence spectroscopy and scanning tunneling microscopy