Circumvention of interference by sulfhydryl compounds in azo dye determination of. beta. -naphthylamine from synthetic protease substrates
In attempting to measure the activity of benzoyl-arginine-..beta..-naphthylamide hydrolase on the synthetic substrate benzoyl-DL-arginine-..beta..-naphthylamide, we observed that L-cysteine, used as a sulfhydryl activator, and four other sulfhydryl compounds, viz., dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, and glutathione, interfered seriously with the subsequent reaction of ..beta..-naphthylamine and naphthanil diazo blue B. It has been found that ..beta..-naphthylamine is quantitatively extractable from the enzyme reaction mixture by a single treatment with ethyl acetate. An aliquot of the ethyl acetate extract appropriately diluted with ethanol and water reacts quantitatively and rapidly with the diazo chromogen. In analyses carried out in this manner, cysteine, mercaptoethylamine, and glutathione up to 20 mM no longer interfered in the azo dye formation; interference by dithiothreitol or mercaptoethand was significantly minimized.
- Research Organization:
- Univ. of Rochester, NY
- OSTI ID:
- 7128029
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 70; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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