Recombinant Human BLMH/Bleomycin Hydrolase Protein, CF

Catalog #: 6200-CY Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6200-CY-010

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Recombinant Human BLMH/Bleomycin Hydrolase Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to hydrolyze Met-AMC. The specific activity is >500 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human BLMH/Bleomycin Hydrolase protein
Ser2-Glu455, with an N-terminal Met and 6-His tag

Accession #

Q13867

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

53 kDa

SDS-PAGE

45-50 kDa, reducing conditions

Product Datasheets

6200-CY

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

6200-CY

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. 6 months from date of receipt, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 V_max* (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).

Per Well:

rhBLMH: 0.05 µg
Substrate: 1 mM

Reconstitution Calculator

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.

References

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.