Members of the Sco protein family have been proposed to function in the assembly of cytochrome c oxidase in the respiratory chain of all aerobic life forms. The Sco protein in Bacillus subtilis, BsSco, is characterized for its folding/unfolding behavior in the presence or absence of Cu(II) in this study. The folding/unfolding of apo-BsSco is investigated by CD and fluorescence spectroscopies. BsSco follows an apparent two-state mechanism in both folding and unfolding processes. The two apo forms of BsSco, reduced and oxidized, exhibit similar equilibrium stabilities suggesting that the formation of an intramolecular disulfide in oxidized apo-BsSco does not add to BsSco’s overall stability. In contrast, Cu(II) binding to reduced apo-BsSco results in extreme stabilization and resistance to unfolding in urea. However, when Cu(II) is present with unfolded, reduced apo-BsSco, the protein is rapidly oxidized. Another widely used denaturant, GdnHCl, is able to unfold Cu(II)-BsSco by allowing the loss of Cu(II) from the metal/protein complex. When the presence of Cu(II)-BsSco complex and the protein’s folded state are monitored simultaneously, the unfolding of Cu(II)-bound BsSco occurs coincidently with Cu(II) dissociation. We suggest that the loss of Cu(II) binding and the loss of BsSco’s native conformation are coincident, which leads to the conclusion that Cu(II)-BsSco does not unfold until it forfeits Cu(II). The kinetics of folding/unfolding of reduced, oxidized and Cu(II) bound BsSco are explored by stopped-flow fluorescence spectroscopy. The rate constants at which the two apo forms of BsSco fold and unfold are measured and plotted versus denaturant concentration. Reduced and oxidized forms of apo-BsSco are similar in folding and unfolding kinetics. Cu(II)-involved refolding kinetics of BsSco show that Cu(II) is able to accelerate the rate of refolding. However, the involvement of Cu(II) in the refolding process results in two competing processes: oxidation and Cu(II) binding. Which process predominates depends on the refolding rate which further depends on the denaturant concentration. This study has provided direct evidence for metal-involved stabilization of BsSco which is beneficial to efficiently fulfill its copper trafficking duty in a cellular environment. / Thesis (Master, Biochemistry) -- Queen's University, 2010-12-17 17:24:09.598

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Mercury-contaminated sediments are found in many locations throughout North America and the world. Release of Hg from such sediments and subsequent biological uptake can result in biomagnification in associated ecosystems. This study focused specifically on a stabilization technique involving the addition of reactive media to the sediment matrix to immobilize Hg and reduce its bioavailability. A series of batch and column experiments was conducted over a range of physical and geochemical conditions to evaluate the propensity of a diverse set of reactive media to stabilize Hg in sediment with high organic carbon and clay content. The additives, selected to promote adsorption and precipitation of Hg, included natural attapulgite (palygorskite) clay, organically-modified clay, elemental sulfur, a strong reductant, and mixtures thereof. The results of the batch experiments indicated that addition of reactive media to the sediment led to substantially lower aqueous concentrations of Hg relative to untreated sediment. The stabilization of Hg was observed to be dependent on mass of added reagent, with generally greater treatment observed for the higher masses of reagent evaluated. Aqueous concentrations of Hg were reduced from > 800 ng L-1 in control samples to < 50 ng L-1 in treated samples for all of the reactive media at the highest mass proportions evaluated. The effectiveness of Hg stabilization using the sulfur-based blends was strongly affected by contact with atmospheric oxygen, with better treatment observed in oxygen-limited conditions. The results of the column tests showed that relatively low concentrations of Hg (< 50 ng L-1) were leached from untreated sediment, maintained under anoxic conditions, with steadily percolating water. However, increased concentrations of Hg (> 200 ng L-1) were observed in column effluent collected from the untreated sediment following flow interruption. This release of high concentrations of Hg was not observed for the columns containing treated sediment after stagnation. Particularly low Hg concentrations were maintained in effluent collected from the sulfur-based treatment mixtures (< 40 ng Hg L-1) for the entirety of the experimental duration, regardless of flow perturbations.

Soil structure and crop response under annual compactive loads of 10 and 18 t/axle and compaction alleviation treatments were assessed in a continuous corn production system. The alleviation treatments consisted of chemical conditioning with a non-ionic surfactant, and subsoiling with narrow-tined and winged implements. Parameters utilized for determining effects on soil structure were bulk density, water retention characteristics and soil profile characteristics. Significant topsoil and subsoil compaction resulted from both loading levels. Crop yield reductions due to compactive loading ranged from 18 to 27% under optimal weather conditions and were as high as 55 to 86% under adverse weather conditions. Soil amendments by chemical conditioning had no beneficial effect on soil structure or crop yields. Inter-row subsoiling with narrow-tined and winged subsoilers was effective in reducing soil compaction and increasing crop yields. / Soil bulk density measurements gave good indication of overall compaction to a depth of 0.4 m and correlated well with crop yields, but were unable to locate differences between compactive treatments. Water retention characteristics indicated significant differences between compactive treatments to a depth of 0.6 m. Soil profile characteristics were useful for determining ped type and size as well as rooting depth and gave the best overall assessment of compaction alleviation effects. / Studies were also conducted to develop a method for predicting soil compactibility that would serve to manage soil compaction at the farm level. Preliminary studies focused on the determination of soil material properties for predicting bulk density changes under applied loads. Further investigations were conducted on the variations in constrained modulus, cone index and soil bulk density values under uniaxial loading. A model relating constrained modulus as a function of cone index, bulk density and soil moisture content was developed. Subsequent studies conducted yielded significant relationships between penetration resistance and soil compactibility as indicated by constrained modulus and coefficient of compressibility. Relationships obtained between penetration resistance and parameters of compactive work and bulk density change indicated the feasibility of assessing soil compactibility at the farm level.