Recombinant Human ABP1/AOC1 Protein, CF

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

Recombinant Human ABP1/AOC1 Protein, CF Summary

• Details of Functionality: Measured by its ability to produce hydrogen peroxide during the oxidation of histamine. The specific activity is >40 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human ABP1/AOC1 protein
  Glu20-Val751, with a N-terminal 6-His tag
• Accession #: CAA55046
• N-terminal Sequence: Inconclusive, His predicted. Protein identity confirmed by MS analysis of tryptic fragments.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: AOC1
• Purity: >90%, 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: 84 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: 83-92 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 HEPES, NaCl, Glycerol.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, pH 7.5
  • Recombinant Human ABP1/AOC1 (rhABP1) (Catalog # 8298-AO)
  • Coupling Enzyme: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • Substrate Component 1: Histamine (Sigma, Catalog # H7250), 20 mM stock in deionized water
  • Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhABP1 to 1 ng/µL in Assay Buffer.
  2. Prepare a Substrate Mixture containing 10 µM Histamine, 2 units/mL HRP, and 100 µM AUR in Assay Buffer.
  3. Load 50 µL of 1 ng/µL rhABP1 into wells of a plate and start the reactions by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing Assay Buffer instead of rhABP1.
  4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode. Note: A cutoff must be set manually at a wavelength of 570 nm.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 5 µM Histamine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

  Per Well:
  • rhABP1: 0.05 µg
  • Histamine: 5 µM
  • HRP: 1 unit/mL
  • AUR: 50 µM

Notes

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

Alternate Names for Recombinant Human ABP1/AOC1 Protein, CF

• ABP
• ABP1
• amiloride binding protein 1 (amine oxidase (copper-containing))
• Amiloride-binding protein
• amiloride-sensitive amine oxidase [copper-containing]
• amiloride-sensitive amine oxidase
• AOC1
• AOC1DAOamiloride-binding protein-1
• DAO1
• Diamine Oxidase
• EC 1.4.3
• EC 1.4.3.22
• Histaminase
• KAO
• Kidney amine oxidase

Background

Amiloride binding protein (ABP1), also known as diamine oxidase (DAO) and amine oxidase copper domain-containing protein 1 (AOC1), is an approximately 110 kDa member of the copper-containing amine oxidase family (1, 2). ABP1 is a glycosylated enzyme that forms disulfide-linked homodimers (3). It is stored in cytoplasmic granules of epithelial cells in the kidney, placenta, uterus, lung, and intestine (1, 4), and its extracellular release can be induced by heparin (5). ABP1 exhibits a substrate preference for histamine and putrescine, generating hydrogen peroxide and ammonia in an oxidative deamination reaction (3). ABP1 activity plays an important role in the catabolism of histamine and other bioactive polyamines (6, 7). Deficiencies in ABP1 can result in histaminosis or dietary histamine intolerance (8, 9).

1. Novotny, W.F. et al. (1994) J. Biol. Chem. 269:9921.
2. Barbry, P. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7347.
3. McGrath, A.P. et al. (2009) Biochemistry 48:9810.
4. Liang, X.H. et al. (2010) Endocrinology 151:5007.
5. Klocker, J. et al. (2004) Vascul. Pharmacol. 40:293.
6. Jones, B.L. and G.L. Kearns (2011) Clin. Pharmacol. Ther. 89:189.
7. Bieganski, T. et al. (1983) Biochim. Biophys. Acta 756:196.
8. Sattler, J. and W. Lorenz (1990) J. Neural Transm. Suppl. 32:291.
9. Maintz, L. and N. Novak (2007) Am. J. Clin. Nutr. 85:1185.

Bioinformatics

• Gene Symbol: AOC1
• Uniprot:
  • CAA55046()