Received on August 8, 2008
Accepted on December 19, 2008

General Clinical Chemistry

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

¹ Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, University of Perugia, Perugia, Italy
² Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, University of Perugia, Perugia, Italy, and San Raffaele Telethon Institute for Gene Therapy, Milano, Italy
³ San Raffaele Telethon Institute for Gene Therapy, Milano, Italy

^* To whom correspondence should be addressed. E-mail: orly{at}unipg.it.

BACKGROUND: The determination of cellular -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 -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 -galactocerebrosidase activity can be performed following complete inhibition of -galactosidase activity.

METHODS: We performed the assay using 2–7.5 µg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone--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 (_ex 360 nm, _em 446 nm).

RESULTS: AgNO₃ was a competitive inhibitor of -galactosidase [inhibition constant (K_i) = 0.12 µmol/L] and completely inhibited -galactosidase activity when used at a concentration of 11 µmol/L. Under this condition, the -galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect -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.