Date of Award


Publication Type

Master Thesis

Degree Name



Chemistry and Biochemistry

First Advisor

Mutus, Bulent,


Chemistry, Biochemistry.



Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.


Nitric oxide (NO), a free radical species is an important retrograde messenger in cell regulation, but is cytotoxic in higher concentrations. Superoxide (O$\sb2\sp{{\cdot}-}$) and NO react yielding peroxynitrite anion (OONO$\sp-)$ which is a potent and toxic oxidant that can attack a wide range of biological targets, for eg. in regulation of blood flow, neurotransmission, sepsis etc. In proteins, peroxynitrite mediated damage occurs via nitration of the aromatic side chains. In the present study, we examined the susceptibility of the two Tyr residues of calmodulin (CaM) to OONO$\sp-$ mediated nitration in vitro as indicated by the amino acid analysis. CaM and OONO$\sp-$ modified CaM were compared with respect to the activation of the CaM dependent enzymes calcineurin phosphatase (CaN) and phosphodiesterase (PDE). Upon modification, the estimated K$\sb{\rm activity}$ for CaN was $\sim$60 fold lower than that observed with the native CaM. There were no apparent change in the K$\sb{\rm activity}$ of PDE, but the extent of activation was reduced by $\sim$79% of that of native CaM. We assessed the binding patterns of CaM (bovine and octopus) with a peptide analog of smMLCK which is the CaM binding domain of smMLCK enzyme, but the OONO$\sp-$ modified CaM failed to bind the same peptide. The effect of peroxynitrite modification on the metal binding properties of CaM were probed by aromatic energy transfer Tb$\sp{3+}$-fluorescence spectroscopy. Energy transfer efficiency of a 14 mer peptide containing 3-nitro-Tyr analogous to the 3$\rm\sp{rd}\ Ca\sp{2+}$ binding loop of CaM was diminished by $\sim$3 fold in comparison to the peptide containing Tyr side chain. The Tb$\sp{3+}$ binding data from nitrated CaM, when normalized was sensitive to the occupation of all four Ca$\sp{2+}$ binding sites whereas with native CaM, the occupation of the 3$\sp{\rm rd}$ and 4$\sp{\rm th}$ sites were detected. This suggests that OONO$\sp-$ modification induced a conformational change on CaM.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1997 .P379. Source: Masters Abstracts International, Volume: 37-01, page: 0264. Adviser: Bulent Mutus. Thesis (M.Sc.)--University of Windsor (Canada), 1997.