Fluorimetric assays for N-acetylgalactosamine-6-sulfatase and arylsulfatase B based on the natural substrates for confirmation of mucopolysaccharidoses types IVA and VI.
BACKGROUND
Treatments have been developed for mucopolysaccharidoses IVA (MPS IVA) and MPS VI suggesting the need for eventual newborn screening. Biochemical enzyme assays are important for diagnosis. Previously reported fluorimetric assays of the relevant enzymes are based on substrates with poor activity or specificity.


METHODS
We developed new fluorimetric assays for N-acetylgalactosamine-6-sulfatase (GALNS) and arylsulfatase B (ARSB) based on the natural substrates, N-acetylgalactosamine-6-sulfate (and 4-sulfate), which have improved activity and specificity toward the relevant enzymes. The new substrates were tested on dried blood spots on newborn screening cards, and assays showed acceptable linearity in response with the amount of enzyme present (using quality control samples).


RESULTS
When tested on dried blood spots from random newborns and affected patients, the assays showed good discrimination between the 2 sample groups.


CONCLUSIONS
The analytical range of the new fluorimetric assays, defined as the ratio of enzyme-dependent-to-enzyme-independent assay response, is likely to be insufficient to use these assays for newborn screening. Rather, these new fluorimetric assays should be useful in a diagnostic lab to confirm a diagnosis via biochemical enzyme testing.
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Arun Babu
AB
Kumar
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Zdenek
Z
Spacil
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Farideh
F
Ghomashchi
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Sophia
S
Masi
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8591, Japan.
Tomomi
T
Sumida
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8591, Japan.
Makoto
M
Ito
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Frantisek
F
Turecek
Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.
C Ronald
CR
Scott
Department of Chemistry, University of Washington, Seattle, WA 98195, USA; Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Electronic address: gelb@chem.washington.edu.
Michael H
MH
Gelb
D001688:Biological Products / Q000378:metabolism*; D002811:Chondroitinsulfatases / Q000032:analysis* / Q000378:metabolism; D059788:Dried Blood Spot Testing; D057075:Enzyme Assays; D005470:Fluorometry; D006801:Humans; D007231:Infant, Newborn; D009083:Mucopolysaccharidoses / Q000145:classification / Q000175:diagnosis* / Q000201:enzymology*; D020051:N-Acetylgalactosamine-4-Sulfatase / Q000032:analysis* / Q000378:metabolism; D015997:Neonatal Screening; D011487:Protein Conformation; D013379:Substrate Specificity
D001688:Biological Products; D002811:Chondroitinsulfatases; D020051:N-Acetylgalactosamine-4-Sulfatase; C581941:ARSB protein, human; C102295:GALNS protein, human