Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -70 °C as supplied.
3 months, -70 °C under sterile conditions after opening.
Assay Buffer: 50 mM NaOAc, 100 mM NaCl, 1 mM CaCl2, 0.5% (w/v) Brij-35, pH 5.0
Recombinant Human Proprotein Convertase 2/PCSK2 (rhPCSK2) (Catalog # 6018-SE)
Substrate: L-PYROGlu-Arg-Thr-Lys-Arg-AMC (Catalog # ES013), 4 mM stock in deionized water
F16 Black Maxisorp Plate (Nunc, Catalog # 475515
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhPCSK2 to 0.4 µg/mL in Assay Buffer.
Dilute Substrate to 100 µM in Assay Buffer.
Start the reaction by mixing equal volumes of 0.4 µg/mL rhPCSK2 and 100 µM Substrate together and incubate at 37 °C for 30 minutes. For a Substrate Blank, mix Assay Buffer and Substrate together and incubate at 37 °C for 30 minutes.
Into a plate, load 100 µL of the reaction and Substrate Blank (in triplicate).
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
Incubation time (min) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).
rhPCSK2: 0.02 µg
Substrate: 50 µM
Background: Proprotein Convertase 2/PCSK2
Proprotein Convertase 2 (PCSK2) is a subtilisin-like serine peptidase that processes proteins into biologically active products in the secretory pathway. Like other furin/kexin family members, PCSK2 cleaves its substrates primarily after sites of paired basic amino acid residues (1). Autoactivation of PCSK2 occurs in a post‑Golgi compartment of the secretory system (2). Interaction with the secretory protein 7B2 is required for proper activation of PCSK2 (3). The N-terminal domain of 7B2 stabilizes active PCSK2, and a C-terminal fragment of 7B2 can inhibit PCSK2 activity (4). rhPCSK2 was coexpressed with 7B2 to facilitate activation of the enzyme. PCSK2 is a major proteolytic processing enzyme of the regulated secretory pathway of the neuroendocrine system, where it generates a number of hormones and neuropeptides (5). Products of PCSK2 processing include enkephalins, insulin, somatostatin, dynorphin, and LHRH.
Zhou, A. et al. (1999) J. Biol. Chem. 274:20745.
Lamango, N.S. et al. (1999) Arch. Biochem. Biophys. 362:275.
Zhu, X. and I. Lindberg (1995) J. Cell Biol. 129:1641.
Lamango, N. et al. (1996) Arch. Biochem. Biophys. 330:238.
Rouillé, Y. et al. (1995) Front. Neuroendocrinol. 16:322.
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