Moepya, Refilwe2023-11-172023-11-172022https://hdl.handle.net/10539/37027A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2022Long-term occupational exposure to inorganic arsenic (iAs), through inhalation, skin contact, or ingestion, can induce a wide range of negative health effects, including cancer, neurological disorders, and cardiovascular diseases. Colorimetry, inductively coupled plasma mass spectrometry (ICP-MS), and atomic absorption spectrometry (AAS) are some of the currently used techniques for the detection of arsenic. However, these techniques are expensive, technically challenging and it is difficult to make real-time decisions due to the time that lapses between sample collection, transportation, analysis, and data processing. As a result, affordable and fast arsenic-monitoring systems are highly required. The research presented in this dissertation explores the development of peptides that are derived from arsenic resistance repressor protein (ArsR) as the molecular recognition element of an arsenic monitoring point of care device. This study aims to qualitatively and quantitatively investigate the binding affinity of arsenic to the ArsR derived peptides using the LC-ESI-MS. The ArsR-derived peptides (Pep1-RJM, Pep2-RJM, Pep3-RJM) were synthesized using the solid-phase peptide synthesis strategy based on the Fmoc approach. The synthesized peptides were purified using semi-preparative high-performance liquid chromatography (prep-HPLC) and characterized using liquid chromatography-tandem mass spectrometry (LCMS). The qualitative and quantitative binding activity of the ArsR-derived peptides to the arsenate (iAs(V), arsenite (iAs(III)), dimethylarsenic acid (DMA(V)), and Phenylarsine oxide (PAO(III)) standards was monitored by measuring the difference between the concentration of original unbound forms of the peptide and the concentration of peptide-arsenic complex using a combination of reversed-phase liquid chromatography and electrospray ionization mass spectrometry. The pentavalent arsenic metabolites were successfully reduced to the better binding trivalent state using L-cysteine while the peptides disulphides bond were reduced using a fivefold molar excess of dithiothreitol (DTT). The effect of various parameters such as pH, temperature, initial concentration of As(III) metabolites, reaction time, and peptide concentration on the binding efficiency was investigated. Optimum As(III) binding was observed at pH8, at a temperature of 40 °C, an initial As(III) concentration of 60 µM, a binding reaction time of 15 minutes, and a peptide dosage of 25 µM. The condensation reaction of Pep1-RJM with arsenic showed a fixed 1:1 coordination without the formation of clusters even at higher molar concentrations of the As(III). The equilibrium constant for the interaction of Pep1-RJM with iAs(III), DMA(III), and PAO(III) that leads to the formation of covalent arsenic-sulphur bonds (As-S) was successfully measured using the peak areas of the extracted ion chromatograms of the LC-ESI-MS. The following order of binding affinities was obtained from the LC-ESI-MS: pep1-PAO (4.11 x 108 M-1) > pep1- iAs (3.47 x 108 M-1 )> pep1-DMA ( 1.68 x 108 M-1 ). Findings suggest that thiol-containing peptides derived from the ArsR protein are capable of effectively binding arsenic in urine and the LC-ESI-MS is an efficient tool in the quantification of arsenic binding.enInorganic arsenic (iAs)ArsR-derived peptidesArsenic-binding peptidesDissertation