PMID: 2119806Sep 19, 1990Paper

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli

Biochimica Et Biophysica Acta
M van de KampG W Canters

Abstract

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence dec...Continue Reading

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Citations

Jul 12, 2001·Chemistry : a European Journal·I M van AmsterdamG W Canters
Feb 6, 2003·International Journal of Biological Macromolecules·R GuzziL Sportelli
Mar 17, 2011·Journal of the American Chemical Society·Kyle M LancasterHarry B Gray
May 29, 2000·European Journal of Biochemistry·O FarverI Pecht
Dec 5, 2009·Applied Magnetic Resonance·Silvia SottiniEdgar J J Groenen
Feb 12, 2009·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Ana-Maria SevcencoPeter-Leon Hagedoorn
Aug 31, 2010·Archives of Biochemistry and Biophysics·Chao XuBaolu Zhao
Sep 12, 2009·Journal of Magnetic Resonance·H BlokJ Schmidt
Mar 1, 2011·The FEBS Journal·Sharmini AlagaratnamGerard W Canters
Jan 25, 2006·Analytical Biochemistry·Sofya KuznetsovaGerard W Canters
Aug 12, 2014·Biosensors & Bioelectronics·Namik AkkilicNusrat J M Sanghamitra
Oct 8, 1999·Biospectroscopy·U KolczakG W Canters
Jul 1, 1997·European Journal of Biochemistry·G Van PouderoyenG W Canters
Sep 1, 1996·European Journal of Biochemistry·S J KroesG W Canters
Jun 10, 2003·Journal of Mass Spectrometry : JMS·Vincenzo CunsoloM Ph Verbeet
May 10, 2006·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Maria FittipaldiEdgar J J Groenen
Feb 2, 2018·Scientific Reports·Anju YadavSri Rama Koti Ainavarapu
Sep 18, 2007·Chembiochem : a European Journal of Chemical Biology·Gaetano D ManettoCarmelo La Rosa
Sep 15, 2005·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Ralf SchmauderThijs J Aartsma
Jul 2, 2014·The Journal of Biological Chemistry·Anthony LeymeMikel Garcia-Marcos
Jul 29, 2010·Protein Science : a Publication of the Protein Society·Wesley B Asher, Kara L Bren
Jun 28, 1993·FEBS Letters·G W Canters, G Gilardi

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