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Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii
- Source :
- European Journal of Biochemistry. 214:475-481
- Publication Year :
- 1993
- Publisher :
- Wiley, 1993.
-
Abstract
- Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five column-chromatography steps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100-fold, resulting in a specific activity for H2 evolution of 935 mumol.min-1.mg protein-1 at 25 degrees C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60 degrees C, the optimal pH value is 6.9. The Km value for methyl viologen is 0.83 mM, for ferredoxin, 35 microM. From SDS/PAGE gels, the protein was judged to be pure. On non-denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 kDa. If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4 Fe atoms/mol. The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.
- Subjects :
- Hydrogenase
Stereochemistry
Iron
Molecular Sequence Data
Chlamydomonas reinhardtii
Electron donor
Biology
Biochemistry
chemistry.chemical_compound
Animals
Amino Acid Sequence
Anaerobiosis
Amino Acids
Peptide sequence
Ferredoxin
chemistry.chemical_classification
Molecular mass
Temperature
Hydrogen-Ion Concentration
biology.organism_classification
Adaptation, Physiological
Enzyme
chemistry
Electrophoresis, Polyacrylamide Gel
Specific activity
Hydrogen
Subjects
Details
- ISSN :
- 14321033 and 00142956
- Volume :
- 214
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry
- Accession number :
- edsair.doi.dedup.....f189efd42932b7fdf2041cf1c1c25471