A long-wavelength fluorescent substrate for continuous fluorometric determination of a-mannosidase activity: Resorufin a-d-mannopyranoside

• Published in: Analytical biochemistry Vol. 399; no. 1; pp. 7 - 12
• Main Authors: Coleman, Daniel J, Kuntz, Douglas A, Venkatesan, Meenakshi, Cook, Gabriele M, Williamson, Staci P, Rose, David R, Naleway, John J
• Format: Journal Article
• Language: English
• Published: 01.04.2010
• Subjects: Drosophila
• ISSN: 0003-2697
• DOI: 10.1016/j.ab.2009.11.039

A simple and reliable continuous assay for measurement of a-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin a-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK sub(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi a-mannosidases. The assay performed using recombinant Drosophila Golgi a-mannosidase (dGMII) has been shown to give the kinetic parameters K sub(m) of 200 kM and V sub(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known a-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.