CHARACTERIZATION OF A NEW HYALURONIC ACID N-DEACETYLASE

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hyaluronic acid (HA) N-deacetylase, the enzyme that removes N-bonded acetyl groups from skin HA in the 7th decade of life (Longas et al., 1987, Carbohydr. Res. 159:127-136), is specific for N-bonded acetyl moieties of D-GlcNAc and D-GalNAc where C1 is beta-linked (Longas et al., 2003, BioMacromolecules 4:189-192). preliminary results have provided partial characterization of HA N-deacetylase in skin homogenate of 76.5 [unreadable] 1.12 year-old subjects, using fractional precipitation with (NH4)2SO4, and polyacrylamide gel electrophoresis, under denaturing and nondenaturing conditions in 1- and 2-dimensions. Enzyme activity was established after incubation of each protein-containing gel (protein-gel) with a 5% (w/v) polyacrylamide gel containing 0.013% (w/v) HA (HA-gel). The protein-gel and the HA-gel were superimposed, and incubated at 37oC for 12 hr in a moist chamber of 0.05 M (NH4)2SO4 buffer (pH 7.86). The protein-gel was stained for protein in Coomassie blue, and the HA-gel was stained for white fluorescence in 0.01 % (w/v) Calcofluor white M2R/0.50 M NaCl. After the sandwich reaction, the HA-gel that reacted with nondenatured homogenate revealed three fluorescent bands, but the one that reacted with denatured homogenate revealed one. The white fluorescence on the HA-gels should originate from N-deacetylated HA (Trudel et al.,1990, Anal. Biochem. 189:249-253). Therefore, the data show that HA-N-deacetylase(s) is composed of three isozymes of apparent molecular weights 79,000;72,000;and 63,000 Daltons, with the smallest one retaining biologic activity after denaturation. Mass Spectrometry is now being employed to more fully characterize these protein(s). After the first round of measurements, the investigators decided to try a new isolation method to minimize contamination.