The acid-catalyzed inversion of sucrose is often taken as an example of a first order reaction. It is, however, influenced by many factors such as temperature, type of acid used, concentration of sucrose, and the concentration of acid. What has received little attention so far is the influence of addition, in particular, other salts to the reacting solution. In this study, the influence of different salt solutions on the kinetics of sucrose inversion rate was studied at 29 °C by use of optical rotation measurements. The salts chosen for this study are readily soluble in sucrose solution and they provide an opportunity to study the interaction of electrolytes in aqueous solution of sucrose as well as their effects on the inversion of sucrose kinetics. The rates are found to be influenced by the concentration of the salts. No significant differences was measured when the salt were dissolved either in the sucrose or in the acid solutions. The influence of added salts to saccharide solutions was determined by evaluating the difference between the rotation of pure saccharides solutions and the rotation of pure saccharide solutions with salts. The changes in optical rotation were compared to the Hofmeister series. The saccharide-salt systems containing acidic salts (Na2HPO4 or NaH2PO4) were found to be dependent on the pH. Changing the molar ratio of sucrose and salt added also had an influence of the change in optical rotation. / Chemistry / M. Sc. (Chemistry)

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

When solutions of various metal 8-quinolinolates or beta-diketonates in an organic solvent were contacted with an aqueous phase and vigorously stirred to generate a large interfacial area, a reversible decrease in the organic phase concentration of the complex was observed. The magnitude of this decrease varied with interfacial area, solvent, temperature, and the nature and concentration of the complex. Analysis of the phenomenon using the Langmuir isotherm showed that the concentration change may be explained by adsorption of significant quantities of the complexes at the increased liquid-liquid interface generated by stirring. Such adsorption was found to complicate extraction kinetics measurements using the high-speed stirring technique when the product chelate is interfacially active, distorting the absorbance/time profile from which rate constants are derived, altering the interfacial area in the reaction vessel, and displacing reactant molecules from the interface. Neutral surfactants were observed to have similar effects. Chelate adsorption was also demonstrated to affect metal ion extraction equilibria, shifting the pH 1/2 value associated with a given metal ion. The magnitude of this shift was found to depend on the concentration of the chelate, its interfacial adsorption constant, and interfacial area. Differences in the pH 1/2 shift were shown to serve as a means of separating metal ions. Studies of the rate of nickel extraction by 8-quinolinols showed that the distribution constant and interfacial activity of the ligand are important factors governing the balance between bulk and interfacial pathways in the extraction. The interfacial rate constant for a given ligand was independent of organic solvent and was typically 10 times larger than the corresponding bulk value, indicating that the interface, although essentially aqueous in character, is a more conducive medium for the reaction of the metal ion and ligand.

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast cytochrome c peroxidase and the cytochrome c from horse, tuna, and yeast, including several site-specific mutants of either the cytochrome c or cytochrome c peroxidase, have been studied. The orientations of the various cytochromes c within electrostatically-stabilized complexes with the peroxidase are not equivalent. This is shown by differential decreases in the rate constants for cytochrome reduction by neutral flavin semiquinones upon complexation which are in the order: tuna ≫ horse > yeast iso-2 > yeast iso-1. We have also directly measured the physiologically-significant intracomplex one-electron transfer rate constants from the ferrous cytochromes c to the peroxide-oxidized species of the peroxidase at several ionic strengths. The rate constants at low ionic strength are highly species dependent, again consistent with the contention that the orientations of the various cytochromes within the complex with CcP are not the same. Increasing the ionic strength in all cases resulted in an increase in the rate constant for the first-order process which controls electron transfer from cytochrome c to the peroxidase Compound I species of the peroxidase. When the two proteins are immobilized by covalent cross-linking, no such rate enhancement is observed, suggesting that the ionic strength effect is manifested by an increase in the number of geometric orientations between the two proteins which results in more rapid electron transfer. Similar rate enhancing effects are observed when positively charged residues on the surface of cytochrome c are converted to electrostatically neutral amino acids by site-specific mutagenesis. The effect of site-specific mutagenesis of two residues of cytochrome c peroxidase have also been studied. His-181, when converted to a glycine has little effect on the electron transfer rate constant, whereas when Trp-191 is converted to a phenylalanine no intracomplex electron transfer could be observed, indicating an obligatory role of this residue in the electron transfer process.

The gas-liquid reaction between chlorodifluoromethane (R-22) and methanol, in the presence of sodium hydroxide, was investigated in an isothermal, stirred, semi-batch reactor. The objective of the study was to develop a model for the reaction and to identify the kinetic parameters. Reactor temperature was varied from 283 to 303 K, with inlet R-22 partial pressures between 40.5 and 60.8 kPa (absolute). Solutions containing sodium hydroxide concentrations of between 1.5 and 2.5 mol·dm-3 were charged into the reactor prior to each experiment. Preliminary investigations using the R-22-methanol system revealed that stainless steel was an inappropriate choice of material for the reactor as it displayed catalytic tendencies toward trimethyl orthoformate formation. Consequently, the reactor was constructed from glass and was equipped with an internal cooling coil, a single heating jacket and a temperature control unit. Liquid samples that were withdrawn from the reactor were degassed under vacuum to remove residual chlorodifluoromethane, and thereby inhibit further reaction. Spectrophotometry was used to analyze the liquid samples to determine the concentration of chloride ions in solution. The products obtained were difluorodimethyl ether (major product) and trimethyl orthoformate (by-product) as well as sodium chloride and sodium fluoride salts. Difluorodimethyl ether is a potential replacement for ozone depleting CFC refrigerants. A Box-Behnken experimental design was used to investigate the effect of reaction conditions on the product distribution. Variations in the reaction temperature, initial concentration of sodium hydroxide and inlet partial pressure of R-22 were considered. The modeling of the gas-liquid reactions was based on the -dehydrohalogenation mechanism. Since gas solubility in a liquid decreases in the presence of dissolved salts, the "salting-out" effect on mass transfer was included in the reactor model. Sechenov coefficients for sodium chloride and sodium fluoride were combined to give a salt Sechenov coefficient Ksalt . It was known from the literature that the presence of precipitated salts causes inefficient mixing and inhibits mass transfer, particularly in this system due to the relatively low salt solubilities in methanol. This mixing effect was also included in the appropriate mass transfer terms of the reactor model. The experimental data was fitted to a proposed kinetic scheme. Kinetic parameters for each of the proposed reactions, the Sechenov ‘salting out’ coefficients and the mixing parameter were obtained through the use of a non-linear, least-squares optimization algorithm. For the kinetic study, activation energies of 89.12 and 45.83 kJ·mol-1 were obtained for the difluorodimethyl ether and trimethyl orthoformate formation reactions, respectively, with a Sechenov salt coefficient of 0.712 and a mixing parameter of 22.43. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2013.

Genetic information is encoded in the long sequence of bases which form DNA, which is replicated during cell division by enzymes known as DNA Polymerases. Polymerases replicate DNA extremely accurately to avoid errors which can cause cell death and diseases such as cancer, although the mechanisms behind these extraordinary fidelities are not well understood. A large conformational change in the protein, in which the “fingers" subdomain closes around an incoming nucleotide, is thought to be implicated in these fidelity mechanisms. Here we present an assay to monitor this conformational change in single polymerase molecules, in real-time. We achieve this using total-internal-reflection-fluorescence (TIRF) microscopy to monitor the fluorescence resonance energy transfer (FRET) of an intra-protein dye labelled DNA Polymerase I (KF) as it binds to surface-immobilised DNA. Initially, we investigated the polymerase fingers-conformations during the pre-chemistry polymerisation reaction, resolving forward and backward rates which would be challenging to observe using ensemble techniques. These observations confirmed that KF closes rapidly around complementary nucleotide, but we discovered that the reverse step, fingers-opening, is particularly slow relative to chemistry. These finger kinetics act to remove the influence of the reaction rate-limiting step on fidelity, surprising given decades of investigations have focused on the rate-limiting step as the key determinant of fidelity. We also use our kinetic measurements to quantify contributions of different reaction steps to the macroscopic error rate of the polymerase. Subsequently, we developed our assay to investigate the fingers-conformations across the entire DNA polymerisation reaction. We observed single-nucleotide incorporations, and processive DNA polymerisation at high and low nucleotide concentrations, which suggested heterogeneous nucleotide incorporation rates. The observations demonstrated that the post-chemistry slow step that limits processive polymerisation occurs before post-chemistry fingers-opening, or is accounted for by post-chemistry fingers-opening. We observe a correlation in turn-over kinetics and binary complex kinetics, suggesting that turn-over rates could be limited by the intrinsic dynamics of the binary complex, as seen in other protein systems, although more work is needed on this.

From a review of the published work in the field of cycloadditions, it is evident that further research is needed to establish the mechanism of ketene cycloadditions. This work was initiated with the intent of obtaining kinetic data which will contribute to the elucidation of the mechanism of ketene cycloadditions.

The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.