Specific Determination of {beta}-Galactocerebrosidase Activity via Competitive Inhibition of {beta}-Galactosidase

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 55; no. 3; p. 541
• Main Authors: Martino, Sabata, Tiribuzi, Roberto, Tortori, Andrea, Conti, Daniele, Visigalli, Ilaria, Lattanzi, Annalisa, Biffi, Alessandra, Gritti, Angela, Orlacchio, Aldo
• Format: Journal Article
• Language: English
• Published: Washington Am Assoc Clin Chem 01.03.2009; Oxford University Press
• Subjects: Brain; Disease; Enzymatic activity; Gene therapy; Genetic disorders; Health hazards; Mortality; Proteins; Stem cells; Tissues
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2008.115873

The determination of cellular β-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for β-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of β-galactocerebrosidase activity can be performed following complete inhibition of β-galactosidase activity. We performed the assay using 2-7.5 µg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-β-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO^sub 3^. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (λ^sub ex^ 360 nm, λ^sub em^ 446 nm). AgNO^sub 3^ was a competitive inhibitor of β-galactosidase [inhibition constant (K^sub i^) = 0.12 µmol/L] and completely inhibited β-galactosidase activity when used at a concentration of 11 µmol/L. Under this condition, the β-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect β-galactocerebrosidase activity in as little as 2 µg cell protein extract or 7.5 µg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC^sup -/-^ hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.