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Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

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Abstract

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.

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Acknowledgments

This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute.

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Authors and Affiliations

  1. NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD, 20892-3370, USA

    Omid Motabar, Ke Liu, Noel Southall, Christopher P. Austin & Wei Zheng

  2. Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892-3708, USA

    Omid Motabar, Ehud Goldin & Ellen Sidransky

  3. Imaging Probe Development Center, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892-3708, USA

    Zhen-Dan Shi & Gary L. Griffiths

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  1. Zhen-Dan Shi
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  2. Omid Motabar
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  3. Ehud Goldin
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  4. Ke Liu
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  5. Noel Southall
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  6. Ellen Sidransky
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  7. Christopher P. Austin
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  8. Gary L. Griffiths
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  9. Wei Zheng
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Corresponding author

Correspondence to Wei Zheng.

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Zhen-Dan Shi and Omid Motabar contributed equally to this work

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Shi, ZD., Motabar, O., Goldin, E. et al. Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase. Anal Bioanal Chem 394, 1903–1909 (2009). https://doi.org/10.1007/s00216-009-2879-5

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  • DOI: https://doi.org/10.1007/s00216-009-2879-5

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