J Integr Plant Biol. ›› 2007, Vol. 49 ›› Issue (3): -.DOI: 10.1111/j.1744-7909.2007.00372.x

• Development & Photosynthesis •    

Purification and Characterization of a New Heme-Binding Protein (HBP59) from the Mutant Strain DJ35 of Azotobacter vinelandii

Shao-Min Bian, Huang-Ping Wang, Hui-Na Zhou, Ying Zhao, Jian-Feng Zhao and Ju-Fu Huang   

Abstract: A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Lipmann. After analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the protein was identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra (ABS) in the reduced state exhibited three peaks at 421, 517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichroism (CD) spectrum of HBP59 in the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the De (CD extinction coefficient) values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, ?.65 and ?.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully interacted with heme, its maximal ABS value and Soret CD intensity were increased by approximately 10-fold compared with values before interaction. Therefore, it seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be involved in heme utilization or adhesion.

Key words: absorption spectra, Azotobacter vinelandii, characterization by matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF), circular dichroism spectra and titration, heme-binding protein (H

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