This paper presents a new assay to determine the activity of the lysosomal enzyme α-N-acetylgalactosaminidase (Naga, EC 3.2.1.49) in human serum. It is based on the use of a new chromogenic substrate, DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-D-galactosaminide) and is performed at pH 4.3 and 37 °C. This allows continuous monitoring of the absorbance of the released DNP. The assay can be performed with a standard spectrophotometer. Compared to established methods using an endpoint assay with MU-α-GalNAc (4-methylumbelliferyl-GalNAc), the present method gives a ca. 3-fold higher specific activity, while only one tenth of the serum concentration in the assay is required. Hence, the assay is at least 30-fold more sensitive than that with MU-α-GalNAc. The pH dependence of the reaction with DNP-α-GalNAc in the pH 3.5 to 6.5 region, while using 4% serum in the assay, shows only one peak around pH 4. This pH optimum is similar to that reported with MU-α-GalNAc. In the accompanying paper (Albracht and Van Pelt (2017) Multiple exo-glycosidases in human serum as detected with the substrate DNP-α-GalNAc. II. Three α-N-acetylgalactosaminidase-like activities in the pH 5 to 8 region. Biochim. Biophys. Acta 159 (2017) Part I and II), the method is used to show that, under special assay conditions, three more Naga-like activities can be uncovered in human serum.
Keywords: A380, optical absorbance at 380 nm; DMF, dimethylformamide; DMSO, dimethylsulphoxide; DNP-α-GalNAc; DNP-α-GalNAc, 2,4-dinitrophenyl-N-acetyl-α-D-galactosaminide; DNPH, 2,4-dinitrophenol; DNP−, 2,4-dinitrophenolate; Gla, α-galactosidase A; Lysosomes; MU, 4-methylumbelliferone; Naga; Naga, α-N-acetylgalactosaminidase; New assay; RT, room temperature; S.A., specific activity in nmol substrate per min per mL serum (nmol·min− 1·mL− 1), using 2 mM DNP-α-GalNAc; Schindler disease; pNP-α-GalNAc, para-nitrophenyl-α-GalNAc; α-GalNAc, N-acetyl-α-D-galactosaminide; α-N-acetylgalactosaminidase.