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Related Experiment Videos

A continuous fluorescence assay for sulfhydryl oxidase.

Sonali Raje1, Nicole M Glynn, Colin Thorpe

  • 1Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.

Analytical Biochemistry
|August 31, 2002
PubMed
Summary

A new fluorescence assay detects flavin-dependent sulfhydryl oxidases, crucial proteins involved in cellular processes. This sensitive method quantifies enzyme activity in various biological samples, including human tears.

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Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Flavin-dependent sulfhydryl oxidases are a newly identified protein family.
  • These enzymes catalyze cysteine oxidation to disulfides, reducing oxygen to hydrogen peroxide.

Purpose of the Study:

  • To develop a sensitive fluorescence assay for flavin-dependent sulfhydryl oxidases.
  • To characterize the assay's performance and applicability to biological samples.

Main Methods:

  • Utilized a continuous fluorescence assay with homovanillic acid as the fluorogen.
  • Employed specific thiol concentrations and high horseradish peroxidase levels to overcome assay interferences.
  • Tested purified avian sulfhydryl oxidase and crude chicken egg white.

Main Results:

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  • The assay detected as little as 15 femtomoles of avian sulfhydryl oxidase.
  • Enzyme activity measurements showed linear dependence on enzyme concentration up to 6 nM.
  • Sulfhydryl oxidase activity was detected in various secretory fluids, including human tears.

    • Conclusions:

    • A sensitive and continuous fluorescence assay for sulfhydryl oxidases was successfully developed.
    • The assay is applicable to biological samples, demonstrating activity in human tears.
    • Further research is needed to optimize the assay and understand enzyme interactions.