Investigating Arsenic−Protein Interactions Using Mass Spectrometry: From Hemoglobin to p53

• Author / Creator

  Popowich, Aleksandra

• Chronic exposure to arsenic from groundwater is one of the greatest public health concerns in the world, putting over 170 million people at risk of arsenic-related diseases, including cancer, diabetes, and cardiovascular disease. Although our understanding of the mechanisms by which arsenic induces these biological effects remains limited and unclear, it is known that the binding of arsenic to the thiol groups (R−SH) of cysteine residues in proteins is a contributing factor. The binding of arsenic to proteins may alter their conformation and stability and inhibit their function. This could impede the recruitment of and interaction with other proteins and DNA, resulting in unrepaired DNA and carcinogenesis. Studying the interactions between arsenic and proteins is indispensable for understanding how arsenic affects the body.
  A method combining size-exclusion high performance liquid chromatography (SE-HPLC) with inductively coupled plasma mass spectrometry (ICPMS) that enables the separation and detection of protein-bound arsenic and free arsenic was developed. Simultaneous detection of arsenic and sulfur oxides (from cysteine and methionine residues present in proteins) provided supporting information for the identification of the arsenic−protein complex. The quantitative ICPMS detection allowed for the determination of apparent binding constants. Using this method, the interactions between trivalent arsenicals and four proteins, hemoglobin, peroxiredoxin-1, high mobility group box 1 protein (HMGB1), and p53, have been characterized.
  Investigating the interaction between arsenic and hemoglobin from seven animal species (cow, guinea pig, human, mouse, pig, rabbit, and rat) aimed to address the interspecies differences seen in arsenic distribution, retention in the blood, and toxicity. Results demonstrated that rat hemoglobin generally has the highest affinity for trivalent arsenic of all the species studies.
  Studying the interactions between arsenic and p53 proteins aimed to determine how known amino acid mutations impact the ability of the protein to bind to transcription factor DNA. The developed HPLC-ICPMS method confirmed the binding of small amounts of PAOIII and MMAIII to wild type p53. The interaction between wild type or mutant p53 and its transcription factor DNA was investigated using an enzyme-linked immunosorbent assay (ELISA) to monitor the capabilities of the p53 proteins to bind DNA. The ability of various trivalent arsenicals to inhibit the interaction between p53 and DNA was studied by performing the assay in the presence or absence of arsenic, providing further insight into the possible mechanism of arsenic-induced carcinogenesis.

• Subjects / Keywords

  • p53
  • mass spectrometry
  • arsenic
  • ICPMS
  • protein
  • HPLC
  • hemoglobin

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3P844B9T

• License

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