Multiple exo-glycosidases in human serum as detected with the substrate DNP- α-GalNAc. I. A new assay for lysosomal α-N-acetylgalactosaminidase

Publication date: Available online 7 October 2017 Source:BBA Clinical Author(s): Simon P.J. Albracht, Erik Allon, Johannes van Pelt 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 pH4.3 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 pH3.5 to 6.5 region, while using 4% serum in the assay, shows only one peak around pH4. 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 pH5 to 8 region. Biochim. Biophys. Acta # (2017) #-#), the method is used to show that, under special assay conditions, three more Naga-like activities can be uncovered in human serum.

Source: BBA Clinical - October 7, 2017 Category: Biochemistry Source Type: research