Recombinant Human Dopamine beta-Hydroxylase Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Dopamine beta-Hydroxylase Protein, CF Summary

• Details of Functionality: Measured by its ability to convert tyramine to octopamine. The specific activity is >7,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Dopamine beta-Hydroxylase protein
  Met1-Gly617, with a C-terminal 6-His tag
• Accession #: P09172
• N-terminal Sequence: Leu37 & Ser40
• Protein/Peptide Type: Recombinant Enzymes
• Gene: DBH
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 66 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 65-85 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.1 M NaOAc, 0.5 µM CuCl₂, pH 5.0
  • Recombinant Human Dopamine beta ‑Hydroxylase (rhDBH) (Catalog # 7376-AO)
  • Substrate Component 1: Tyramine (Sigma, Catalog # T2879), 100 mM stock in deionized water
  • Substrate Component 2: N,N-Dimethyl-p-phenylenediamine (DMPD) (Sigma, Catalog # D4139), 500 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhDBH to 1 ng/µL in Assay Buffer.
  2. Dilute both substrate components to 40 mM respectively in Assay Buffer.
  3. Combine equivalent volumes of substrate components for substrate mixture.
  4. Load 50 µL of the diluted rhDBH into a clear plate, and start the reaction by adding 50 µL of the Substrate mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate Mixture without any rhDBH.
  5. Read in kinetic mode for 5 minutes at an absorbance of 515 nm.
  6. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 5200 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhDBH: 0.050 μg
  • Tyramine: 10 mM
  • DMPD: 10 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Dopamine beta-Hydroxylase Protein, CF

• DBH
• DBM
• dopamine beta-hydroxylase (dopamine beta-monooxygenase)
• Dopamine betaHydroxylase
• Dopamine beta-Hydroxylase
• Dopamine beta-monooxygenase
• DOPBHY
• EC 1.14.17.1

Background

Dopamine beta -Hydroxylase (DBH), also known as dopamine beta -monooxygenase, belongs to the copper type II, ascorbate-dependent monooxygenase family. DBH is found within the neurosecretory vesicles of adrenal medullae and the large dense‑cored synaptic vesicles of the sympathetic nervous system as both membrane‑associated and soluble forms (1, 2). It catalyzes the conversion of dopamine to noradrenaline in sympathetic neurons, making it an important enzyme for catecholamine biosynthesis (3, 4). Mutations in the DBH gene that result in low DBH activity are a cause of noradrenaline deficiency (5). This recombinant human DBH is the secreted, soluble form.

1. Dhawan, S. et al. (1987) J. Biol. Chem. 262:1869.
2. Saxena, A. and P.J. Fleming (1983) J. Biol. Chem. 258:4147.
3. Levin, E.Y. et al. (1960) J. Biol. Chem. 235:2080.
4. Friedman, S. and S. Kaufman (1965) J. Biol. Chem. 240:4763.
5. Kim, C.H. et al. (2002) Am. J. Med. Genet. 108:140.

Bioinformatics

• Gene Symbol: DBH
• Uniprot:
  • P09172()