A continuous spectrophotometric assay for arylsulfatase activity, dependent on the formation of complexes between cupric ions and nitrocatechols

✍ Scribed by Alan G. Clark; David A. Jowett; John N. Smith

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 822 KB
• Volume: 118
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90184-6

✦ Synopsis

The nitrocatechols 2-hydroxy-5-nitrophenol and 2-hydroxy-5-methyl-4-nitrophenol formed strongly colored complexes with cupric ions, the dissociation constants of these complexes being 1.61 and 1.74 X 10V4 M, respectively. Complex formation with 2-hydroxy-5-methyl-4nitrophenol was independent of pH between pH 5.4 and 7.2. The aryl-sulfate sulfohydrolase (EC 3.1.6.1) from the New Zealand mollusk, Haliotis iris, was not inhibited by low concentrations of cupric ion so that the enzymatic hydrolysis of sulfate esters of the above nitrocatechols could be monitored continuously in the presence of cupric ions, by following the formation of the yellow complexes. Assay methods based on this phenomenon gave results identical with those obtained by the discontinuous method of alkaline development. Rate measurements were linearly related to enzyme concentration whichever assay method was used. At very high pH, cupric ions decreased the intensity of color of the nitrocatechol anions,