Wright, P. John (2003) Analysis of TEMPO-adducts of protein-based radicals by mass spectrometry. PhD thesis, Concordia University.
Heme peroxidases catalyze the one-electron oxidation of a wide variety of organic and inorganic substrates using hydrogen peroxide (H 2 O 2 ) as an oxidant. Use of these enzymes in environmentally sound biotechnological and industrial applications is limited by their stability under harsh conditions such as high H 2 O 2 concentrations and extremes in pH. In of this thesis, a detailed study on the characterization of the low-pH heme-pocket stability of horseradish peroxidase isozyme c (HRPC) is reported. FTIR y(CO) and Soret-CD spectra of HRPC-CO, and Soret absorption of ferric HRPC from two commercial sources were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers. Both HRPC-CO samples exhibit identical pH 7.0 y(CO) bands at 1934 and 1905cm -1 . However, in the pH 3.0 spectrum of sample A , the 1934cm -1 band was dominant while a broad 1969cm -1 band appeared in sample B , which was assigned to solvent-exposed heme. The intensity of this band was greater in citrate than phosphate buffer, but in formate the 1934cm -1 band remained dominant. In the presence of 1mM CaCl 2 , no time- or buffer-anion-dependent conformation changes were detected, revealing that buffer-anion-dependent leaching of the stabilizing-Ca 2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible-protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation which was detected by SDS-PAGE. The second focus of this thesis is the reactive intermediates generated in the HRPC, cytochrome c peroxidase (CCP), and myoglobin (Mb) polypeptides upon their reaction with H 2 O 2 in the absence of donor substrate.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (PhD)|
|Authors:||Wright, P. John|
|Pagination:||xx, 137 leaves : ill. ; 29 cm.|
|Degree Name:||Theses (Ph.D.)|
|Program:||Chemistry and Biochemistry|
|Thesis Supervisor(s):||English, Ann M.|
|Deposited By:||Concordia University Libraries|
|Deposited On:||27 Aug 2009 17:27|
|Last Modified:||08 Dec 2010 15:26|
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