A continuous fluorescence assay for sulfhydryl oxidase.
Flavin-dependent sulfhydryl oxidases represent a newly discovered family of proteins with a range of cellular locations and putative roles. The avian and mammalian proteins can catalyze the direct oxidation of protein cysteine residues to disulfides with the reduction of dioxygen to hydrogen peroxide. Although thiols interfere with the peroxidase-mediated quantitation of hydrogen peroxide, a very sensitive, continuous fluorescence assay of the sulfhydryl oxidases can be devised with careful selection of thiol substrate concentration and fluorogen. Purified avian enzyme (or crude chicken egg white) was used for these experiments. Homovanillic acid was found to be a suitable fluorogen in the presence of 300 microM thiols from either dithiothreitol or reduced ribonuclease A. High concentrations of horseradish peroxidase minimized the effects of contaminating catalase in biological samples. Using fluorescence microcells, the assay could detect 15fmol of avian sulfhydryl oxidase and the rates were linearly dependent on enzyme concentration up to 6nM. Aspects of the interaction among thiols, homovanillic acid, and peroxidase are discussed which limit the sensitivity of the assay and require that care is exercised in the application of this new procedure. Finally, the assay is used to show that there is sulfhydryl oxidase activity in a number of secretory fluids including human tears.
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Sonali
S
Raje
Nicole M
NM
Glynn
Colin
C
Thorpe
D000818:Animals; D030161:Avian Proteins / Q000032:analysis / Q000378:metabolism; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004527:Egg Proteins / Q000032:analysis / Q000378:metabolism; D005453:Fluorescence; D005949:Glucose Oxidase / Q000378:metabolism; D006719:Homovanillic Acid / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis* / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D013997:Time Factors; D050862:Oxidoreductases Acting on Sulfur Group Donors
D030161:Avian Proteins; D004229:Dithiothreitol; D004527:Egg Proteins; D005949:Glucose Oxidase; D006719:Homovanillic Acid; D010088:Oxidoreductases; D012260:Ribonucleases; D050862:Oxidoreductases Acting on Sulfur Group Donors