posted on 2024-11-18, 13:50authored byRajesh Gupta
Electrospray ionisation mass spectrometry (ESI-MS) has been used to study the non-covalent interactions in dsDNA-drug complexes, a protein-protein complex of DNA polymerase III of E. coli and binding specificity of cisplatin in the formation of bifunctional adducts with DNA. ESI conditions have been developed to maximise the detection of dsDNA and drug-DNA complexes in the positive ion mode, with comparable and/or superior results to those achieved previously using negative ions. The gas phase stabilities of complexes of dsDNA with different classes of drugs have been examined. Different classes of drugs exhibit differences in sequence selectivity, modify DNA in various ways and are also known to stabilise or destabilise dsDNA. ESI-MS/MS, desolvation temperature variation, and in-source collision-induced dissociation experiments have been employed to examine the relative stabilities of drug-dsDNA complexes. The binding of complexes of a new class of metallointercalators containing Ru with dsDNA has also been examined by ESI-MS. Conditions were developed for optimal detection of these complexes in the negative ion mode and their stoichiometries and sequence selectivity have been probed. The relative order of binding affinities was found to correlate with solution studies carried out with related ruthenium compounds containing the extended planar ligands dpq, dpqC and dppz. ESI-MS has also been used to supply a snapshot of components of mixtures of two proteins (?)186 and (?) that form a complex in DNA polymerase III. The stability of the (?-?)186 complex has been studied under various solution and instrumental conditions. The behaviour of this complex was consistent with significant contribution of hydrophobic interactions to its stability, and contrasted with the previously reported behaviour of the Tus-Ter complex. In addition, ESI-MS experiments suggested that the (?) subunit is involved in stabilisatioin of (?)186. This is consistent with earlier experiments in which (?) was shown to stabilise (?) against thermal inactivation. Finally the thesis has examined binding specificity of cisplatin in the formation of bifunctional adducts at three well-characterised binding sites on ssDNA. A novel method for better reaction conditions and purification of ssDNA-cisplatin adducts HPLC has been developed. The bifunctional ssDNA-cisplatin adducts at 5-GG-3, 5-AG-3 and 5-GA-3 sites have been characterised by enzymatic digestion followed by ESI mass spectrometry for identification of binding sites. [Note: this abstract contained scientific formulae that would not come across on this form. Please see the 01Front files Abstract for the full details.]
History
Citation
Gupta, Rajesh, Electrospray ionisation mass spectrometry of biomolecular complexes, PhD thesis, Department of Chemistry, University of Wollongong, 2003. http://ro.uow.edu.au/theses/160
Year
2003
Thesis type
Doctoral thesis
Faculty/School
Department of Chemistry
Language
English
Disclaimer
Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.