The streptococcal flavoprotein NADH peroxidase: Purification, analysis of structural and redox properties, and identification of the active-site cysteinyl derivate
The NADH peroxidase of Streptococcus faecalis 10C1, purified to homogeneity, was studied using a variety of structural and spectroscopic techniques. The cofactor content of the enzyme was established using standard techniques, including atomic absorption analyses for the metal content. The native and subunit molecular weights of the protein were obtained through a combination of analytical ultracentrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, and flavin content. Redox properties were studied by spectral titrations with substrates and/or chemical reductants. An essential oxidized cysteinyl derivative within the enzyme was identified through thio titrations of oxidized and reduced enzyme with 5,5{prime}-dithiobis(2-nitrobenzoic acid), reductive alkylation of the enzyme with iodo(2-{sup 14}C)acetamide, and performic acid oxidation of enzyme labelled metabolically with ({sup 35}S)cysteine. Proteolytic digestion of radiolabelled enzyme followed by peptide purification by high performance liquid chromatography and automated Edman degradation yielded amino acid sequences of the cysteine-containing tryptic and chymotryptic peptides. Preliminary mass spectral analysis of the chymotryptic peptide was performed to probe the structure of the oxidized cysteinyl derivative.
- Research Organization:
- Wake Forest Univ., Winston-Salem, NC (USA)
- OSTI ID:
- 7246127
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
Similar Records
Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr
Recombinant rat liver guanidinoacetate methyltransferase: Reactivity and function of sulfhydryl groups
Related Subjects
PEROXIDASES
MOLECULAR STRUCTURE
ABSORPTION SPECTRA
ACETAMIDE
AMINO ACID SEQUENCE
CARBON 14 COMPOUNDS
CHEMICAL COMPOSITION
CYSTEINE
ELECTROPHORESIS
FRACTIONATION
LIQUID COLUMN CHROMATOGRAPHY
NADH2
REDOX POTENTIAL
STREPTOCOCCUS
SULFUR 35
TRACER TECHNIQUES
ULTRACENTRIFUGATION
AMIDES
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CENTRIFUGATION
CHROMATOGRAPHY
COENZYMES
DAYS LIVING RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
RADIOISOTOPES
SEPARATION PROCESSES
SPECTRA
SULFUR ISOTOPES
THIOLS
550201* - Biochemistry- Tracer Techniques