[Truncated] Reactive oxygen and nitrogen species (RONS) are produced by tissues during normal resting metabolism. One major site of RONS production is skeletal muscle, with its rate of RONS production increasing during exercise. There is evidence that the resulting rise in RONS concentrations plays many important physiological roles. One important mechanism through which RONS operate involves the reversible oxidation of exposed protein thiols of the amino acid cysteine. Unfortunately, as reviewed in chapter one, there are many pitfalls with the protein thiol assays currently available, and research into this field has been hampered by a lack of suitable analytical techniques. For this reason, the primary aim of the first study of this thesis (chapter two) is to develop a technique to measure protein thiols in biological tissues in both their reduced and oxidised states.
Chapter two describes a highly sensitive, quantitative labelling technique that measures the level of global and specific protein thiol oxidation in skeletal muscle. The technique involves the labelling of the reduced and oxidised protein thiols within the same complex tissue sample with different fluorescent dyes. The resulting sample is assayed using a 96-well plate fluorimeter, and individual protein bands are separated using SDS PAGE and later identified using mass spectrometry. We show that artefactual oxidation during sample preparation and analysis has the potential to confound results, and techniques to prevent this are described. We tested the technique by analysing the muscles of mdx (dystrophic) and c57 mice, and found the muscles of mdx mice were significantly (p < 0.05) more oxidised (13.1 ± 1.5 % oxidised thiols) than those of c57 mice (8.9 ± 0.7 % oxidised thiols). This technique provides an effective means to measure the extent to which oxidative stress affects the oxidation of protein thiols in biological tissues.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2012|