Recombinant Human Adenosylhomocysteinease/AHCY Protein, CF
Recombinant Human Adenosylhomocysteinease/AHCY Protein, CF Summary
Product Specifications
Ser2-Tyr432, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
6466-AH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM HEPES, 2 mM MgCl2, 1 mM EDTA, pH 7.0
- Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
- Glutathione, reduced (Amresco, Catalog # 399)
- Adenosylhomocysteine (Sigma, Catalog # A9384), 10 mM stock in 10 mM HCl
- Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
- ThioGlo®3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 mM stock in DMSO
- DMSO (Sigma, Catalog # 154938)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Prepare a 10 mM stock of reduced glutathione in deionized water.
- Prepare the standard curve by diluting the 10 mM reduced glutathione (10,000 pmol/µL) to 10 pmol/µL in Assay Buffer and performing six additional ½ serial dilutions.
- Dilute rhAHCY to 0.667 μg/mL in Assay Buffer.
- Dilute adenosylhomocysteine to 1 mM in Assay Buffer.
- Dilute rhADA to 0.24 mg/mL in Assay Buffer.
- Load the microplate as follows:
a. Reactions: 15 µL of 0.667 μg/mL rhAHCY, 15 µL of 0.24 mg/mL rhADA, and 20 µL of 1 mM adenosylhomocysteine
b. Substrate Blanks: 15 µL of Assay Buffer, 15 µL of 0.24 mg/mL rhADA, and 20 µL of 1 mM adenosylhomocysteine
c. Standard Curve: 50 µL per well - Cover microplate and incubate at 37 °C for 30 minutes.
- Dilute ThioGlo® to 100 µM in DMSO.
- After incubation, add 50 µL of 100 µM ThioGlo® to each well.
- Incubate at room temperature for 5 minutes in the dark.
- Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Free thiol produced* |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the reduced glutathione standard curve using linear fitting and adjusted for Substrate Blank.
Per Well:- rhAHCY: 0.010 µg
- Adenosylhomocysteine: 200 µM
- rhADA: 3.6 μg
- ThioGlo®: 50 µM
Reconstitution Calculator
Background: Adenosylhomocysteinase/AHCY
Human S‑Adenosylhomocysteinase (AHCY) is a cytoplasmic tetramer with a tightly bound NAD co‑factor for each subunit (1, 2). It is the only known enzyme to catalyze the breakdown of S‑adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy hydrolysis is a reversible reaction with an equilibrium favoring AdoHcy formation, but hydrolysis prevails under physiological conditions due to the rapid removal of adenosine and homocysteine. Thus, AHCY’s activity in mammals is directly related to homocysteine level, an independent risk factor for vascular disease (3). It also functions as a regulator of biological transmethylation by controlling the concentration of AdoHcy, a potent competitive inhibitor of all S-adenosyl-L-methionine methyltransferases (1). A mutation in the human AHCY results in AHCY deficiency with increase of plasma creatine kinase, methionine, S‑adenosylmethionine and AdoHcy, delay of myelination, myopathy and psychomotor retardation (4, 5).
- Turner, M. A. et al. (2000) Cell Biochem. Biophys. 33:101.
- Takata, Y. et al. (2002) J. Biol. Chem. 277:22670.
- Gellekink, H. et al. (2004) Eur. J. Hum. Genet. 12:942.
- Baric, I. et al. (2004) Proc. Natl. Acad. Sci. USA. 101:4234.
- Fumic, K. et al. (2007) Eur. J.Hum. Genet. 15:347.
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