Recombinant Human PON1 His-tag Fc Chimera Protein, CF

Recombinant Human PON-1 His-tag Fc Chimera (Catalog # 10175-PO) ismeasured by its ability to hydrolyze phenyl acetate. The activity (orange) isover 3-fold higher than the competitor's PON-1 (green).

2 μg/lane of Recombinant Human PON1 His-tag Fc Chimera was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® blue staining, showing a band at ~74 kDa under reducing conditions.

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

Recombinant Human PON1 His-tag Fc Chimera Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze phenyl acetate. The specific activity is >5000 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human PON1 protein Leu16-Leu355 with an N-terminal 6-His tag and a C-terminal Fc tag
• Accession #: P27169
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• 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: 69-79 kDa, under 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 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, CaCl 2 NaCl, and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 5 mM CaCl₂, pH 8.0
  • Deionized Water
  • Recombinant Human PON1 His-tag Fc Chimera (rhPON1) (Catalog 10175-PO)
  • Substrate: Phenyl Acetate (7.88 M) (Sigma, Catalog # 108723)
  • UV Plate (Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPON1 to 10 µg/mL in Assay Buffer.
  2. Dilute Phenyl Acetate to 40 mM in deionized water.
  3. Load 50 µL of 10 µg/mL rhPON1 in a plate, and start the reaction by adding 50 µL of Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate.
  4. Read plate at a wavelength of 270 nm (absorbance) in kinetic mode for 5 minutes.
  5. 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 1310 M-1cm-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD

  Per Well:
  • rhPON1: 0.5 µg
  • Phenyl Acetate: 20 mM

Notes

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

Alternate Names for Recombinant Human PON1 His-tag Fc Chimera Protein, CF

• A-esterase 1
• Aromatic esterase 1
• arylesterase B-type
• EC 3.1.1.2
• EC 3.1.1.81
• EC 3.1.8.1
• ESA
• ESAesterase A
• K-45
• MVCD5
• paraoxonase 1
• PON 1
• PON1
• PONparaoxonase B-type
• serum aryldiakylphosphatase
• Serum aryldialkylphosphatase 1
• serum paraoxonase/arylesterase 1

Background

Serum paraoxonase 1 (PON-1) is a member of the paraoxonase family which has three members: PON1, PON2, PON3. PON-1 is a lactonase (1,2) but has significant promiscuity (2,3) that allows hydrolysis of a variety of substrates including organophosphate triesters (including many pesticides), arylesters, cyclic carbamates, glucuronides and consequently also pharmaceuticals such as statins. PON-1 is a calcium-dependent, secreted, 43 kDa protein with a 6-bladed propeller structure and an active site gorge containing a His-His catalytic dyad (4). It is a homodimer and retains its hydrophobic signal sequence in the N-terminal region which enables its association with HDL, resulting in its localization predominantly in the plasma (5,6). Low serum PON1 and dysfunctional HDL is associated with many diseases with an inflammatory component including diabetes mellitus (7,8), rheumatoid arthritis, hepatic and renal diseases, psoriasis, and macular degeneration (9,10). PON1 shows a variety of atheroprotective properties (6,11,12) by metabolizing inflammatory lipid peroxides (13). PON-1 activity is low in coronary heart disease patients (14) and contributes to an increased risk of a major adverse cardiovascular event (15). PON-1 also protects against toxicity of organophosphorus compounds used as pesticides (16,17) thought to increase risk of neurodegenerative disorders such as Parkinson's (18) and Amyotrophic Lateral Sclerosis (19).

1. Dragonov, D. I. et al. (2005) Lipid Res. 46:1239.
2. Khersonsky, O. et al. (2005) Biochemistry. 44:6371.
3. Ben-David, M. et al. (2012) J. Mol. Biol. 418:181.
4. Harel, M. et al. (2004) Nature Struct. Mol. Biol. 11:412.
5. Mackness, B. et al. (1998) Gen. Pharmac. 31:329.
6. Aviram, M. et al. (2004) Free Radic. Biol. Med. 37:1304.
7. Rozenberg, O. et al. (2008) Free Radic. Biol. Med. 44:1951.
8. Koren-Gluzer, M. et al. (2011) Atherosclerosis. 219:532.
9. Goswami, N. et al. (2009) Clin. Chim. Acta. 410:1.
10. Mahrooz, A. (2016) Curr. Clin. Pharmacol. 11:259.
11. Berrougui, H. et al. (2012) Free Radic. Biol. Med. 52:1372.
12. Aharoni, S. et al. (2013) Atherosclerosis 228:353.
13. Tavori, H. (2011) Free Rad. Biol. Med. 51:234.
14. Wang, M. et al. (2012) DNA Cell. Biol. 31:975.
15. Tang, W. H. W. et al. (2012) Arterioscler. Thromb. Vasc. Biol. 32:2803.
16. Li, W. F. et al. (2000) Pharmacogenetics. 10:767.
17. Shih, D. M. et al. (1998) Nature. 394:284.
18. Ahmed, H. et al. (2017) Biomed. Pharmacother. 90:638.
19. Gagliardi, S. et al. (2013) Neurotox. Res. 23:370.

Bioinformatics

• Uniprot:
  • P27169(Human)