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Cytochrome c peroxidase : its role in nitrosative stress and purification as a GST-fusion protein

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Cytochrome c peroxidase : its role in nitrosative stress and purification as a GST-fusion protein

Bakas, Iolie C (1999) Cytochrome c peroxidase : its role in nitrosative stress and purification as a GST-fusion protein. Masters thesis, Concordia University.

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Abstract

Cytochrome c Peroxidase (CCP) is a hemoprotein found in the intermembrane space of yeast mitochondria. One of its roles is to protect the cell against oxidative stress by reducing mitochondrial H 2 O 2 to H 2 O. Titration of CCP with peroxynitrite [ONOO(H)] formed a species with an absorption spectrum identical to compound I/II at a ONOO(H)/CCP ratio of 1.65 ± 0.10. Titration of the CCP species formed with ferrocytochrome c, gives a ratio of ferrocytochrome c/CCP = 1.87, indicating that the species formed is compound I. An estimate of the second-order rate constant for the reaction of peroxynitrite with CCP at pH 7.0 is 6.9 ± 2.2 \ 10 5 M -1 s -1 . We propose that the end product of the reaction of CCP with peroxynitrite is nitrite and not the NO 2 · radical, since excess nitrite does not induce reduction of compound 1. We conclude that CCP is a protective enzyme against nitrosative as well as peroxidative stress. A silent BamH I mutation was introduced into the mature sequence of CCP gene as well as BamH I and EcoR I restriction sites at its 5 ' - and 3 ' -ends, respectively. Cloning of this DNA into the pGEX 2T vector yields the GST-CCP construct, and expression of this construct in E. coli produces ~530 mg GST-CCP fusion protein per 12 L of culture. Purification of the fusion protein involves a single affinity chromatography step and free CCP is obtained by cleavage of the fusion protein with thrombin. After recrystallization, 60 mg of highly pure enzyme was produced with 61.5% of the expected activity based on heme content. Further investigations are necessary to improve the yield and activity of CCP as obtained using this procedure.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Bakas, Iolie C
Pagination:xii, 137 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (M.Sc.)
Program:Chemistry and Biochemistry
Date:1999
Thesis Supervisor(s):English, Ann M.
ID Code:855
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:14
Last Modified:08 Dec 2010 10:17
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