Abstract
Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.
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Abbreviations
- A:
-
absorbance
- Da:
-
dalton
- EDTA:
-
ethylenediaminetetraacetic acid
- Fd:
-
ferredoxin
- HPLC:
-
high-performance liquid chromatography
- Km:
-
Michaelis constant
- 2-ME:
-
2-mercaptoethanol
- MV:
-
methyl viologen
- PEG:
-
polyethylene glycol
- PMSF:
-
phenylmethylsulfonyl fluoride
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SiR:
-
sulfite reductase
- Tris:
-
tris (hydroxymethyl) aminomethane
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Takahashi, S., Yoshida, Y. & Tamura, G. Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves. J. Plant Res. 109, 45–52 (1996). https://doi.org/10.1007/BF02344286
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DOI: https://doi.org/10.1007/BF02344286