96 Well UV Transparent Plate (Costar, Catalog # 3635)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute Substrate to 5.0 mg/mL in Assay Buffer.
Dilute rHyaluronan Lyase to 2.0 µg/mL in Assay Buffer.
Load into a plate 50 µL of 2.0 µg/mL rHyaluronan Lyase, and start the reaction by adding 50 µL of 5.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 5.0 mg/mL Substrate.
Read plate at 232 nm in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/M
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Using the extinction coefficient 3800 M-1cm-1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Per Well:
rHyluronan Lyase: 0.1 µg
Substrate: 2.5 mg/mL
Background: Hyaluronan Lyase
The hyaluronan lyase from Streptococci agalactiae is highly specific towards hyaluronan (1). However, a thousand fold lower activity towards unsulfated chondroitin sulfate has also been observed (2). The enzyme activity is Ca2+ dependent (3). During digestion the enzyme moves processively along the hyaluronan chains continuously releasing disaccharide units as it travels (1). The recombinant enzyme can be used to digest hyaluronan and study chondroitin sulfate structure (2). Unlike hyaluronidases, hyaluronan lyase digestion of the substrate creates a double bond at the non-reducing terminus of the product; therefore, the digestion progress can be followed by UV spectrometry and the enzyme can be used for hyaluronan quantification. The recombinant enzyme can also be used for drug screening because the enzyme is a major pathogenic factor for Streptococci agalactiae that causes serious, often fatal, neonatal infections (4). The expressed protein corresponds to the mature form of the native protein (3).
Lin, B. et al. (1994) J. Biol. Chem. 269:30113.
Baker, J. et al. (1997) Biochem. J. 327:65.
Akhtar, M. et al. (2006) J. Biol. Chem. 281:28336.
Hynes, W.L. and Walton, S.L. (2000) FEMS Microbiol. Lett. 183:201.
R&D Systems personnel manually curate a database that contains references using R&D Systems products.
The data collected includes not only links to publications in PubMed,
but also provides information about sample types, species, and experimental conditions.
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