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Clinical Chemistry 55: 541-548, 2009. First published January 15, 2009; 10.1373/clinchem.2008.115873
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(Clinical Chemistry. 2009;55:541-548.)
© 2009 American Association for Clinical Chemistry, Inc.

General Clinical Chemistry

Specific Determination of β-Galactocerebrosidase Activity via Competitive Inhibition of β-Galactosidase

Sabata Martino1,3, Roberto Tiribuzi1,3, Andrea Tortori1, Daniele Conti1,2, Ilaria Visigalli2, Annalisa Lattanzi2, Alessandra Biffi2, Angela Gritti2 and Aldo Orlacchio1,a

1 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, University of Perugia, Perugia, Italy; 2 San Raffaele Telethon Institute for Gene Therapy, Milano, Italy.
3 S. Martino and R. Tiribuzi contributed equally to this work.

aAddress correspondence to this author at: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, University of Perugia, Italy. Fax +390755852187; e-mail orly{at}unipg.it.

Background: 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.

Methods: 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 AgNO3. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer ({lambda}ex 360 nm, {lambda}em 446 nm).

Results: AgNO3 was a competitive inhibitor of β-galactosidase [inhibition constant (Ki) = 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–/– hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors.

Conclusions: 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.






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Copyright © 2009 by the American Association for Clinical Chemistry.