Recombinant Human BLMH/Bleomycin Hydrolase Protein, CF Summary
Ser2-Glu455, with an N-terminal Met and 6-His tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.|
|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.
- Assay Buffer: 50 mM HEPES, 5 mM EDTA, 10 mM DTT, pH 7.0
- Recombinant Human BLMH/Bleomycin Hydrolase (rhBLMH) (Catalog # 6200-CY)
- Substrate: Met-AMC (Bachem, Catalog # I-1265), 100 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhBLMH to 1 µg/mL in Assay Buffer.
- Incubate 1 µg/mL rhBLMH at 37 °C for 30 minutes.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 µL of the 1 µg/mL rhBLMH into a black well plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 2 mM Substrate without any rhBLMH.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmoles/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).
- rhBLMH: 0.05 µg
- Substrate: 1 mM
Background: BLMH/Bleomycin Hydrolase
Bleomycin Hydrolase (BLMH) is a cysteine peptidase of the papain superfamily. It is named for its ability to hydrolyze the antitumor agent bleomycin and inactivate it (1). It has a papain-like catalytic triad (Cys-His-Asp) with optimum activity at neutral pH. In mammals it is expressed ubiquitously in all types of tissues and its expression is up‑regulated in many tumors. It is present in the cytoplasm as homohexameric protein of approximately 300 kDa. In addition to its aminopeptidase activity, it has homocysteine-thiolactonase activity. The normal physiological function of BLMH is not clear. BLMH inactivates bleomycin, a glycopeptide anticancer agent, by deaminating it (2). BLMH has been suggested to play a role in the generation of MHC class I-presented peptides (3, 4). Diminished BLMH activity may contribute to the pathology of Alzheimer’s disease (AD) (5, 6). It is inhibited by cysteine protease inhibitors such as N-ethylmaleimide, iodoacetamide, para‑hydroxymercuribenzoate, and E-64.
Joshua-Tor, L. and S. A. Johnson (2004) in Handbook of Proteolytic Enzymes, Barrett, A. J. et al. eds. pp. 1197.
Schwartz, D. R. et al. (1999) Proc. Natl. Acad. Sci. USA, 96:4680.
Kim, E. et al. (2009) J. Immunol. 183:7379.
Towne, C. F. et al. (2007) J. Immunol. 178:6923.
Suszynska, J. et al. (2010) J. Alzheimers Dis. 19:1177.
Lefterov, I. M. et al. (2000) FASEB J. 14:1837.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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