Spectrophotometric assay for vertebrate collagenase |
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| Authors: | H Weingarten J Feder |
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| Affiliation: | 1. Department of Medicinal Chemistry, A.V. Bogatsky Physical-Chemical Institute NAS of Ukraine, 86 Lustdorfskaya Doroga, Odessa, 65080, Ukraine;2. Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, 154 Acad. Zabolotny Str., Kyiv, 03143, Ukraine;3. Laboratory for Molecular Modeling, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, USA;1. National Wine and Grape Industry Centre, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia;2. The Australian Research Council Training Centre for Innovative Wine Production, University of Adelaide, PMB 1, Glen Osmond, Adelaide, SA 5064, Australia;3. CSIRO Agriculture and Food, Locked Bag 2, Glen Osmond, Adelaide, SA 5064, Australia;4. School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia;5. NSW Department of Primary Industries, Wagga Wagga, NSW 2678, Australia;1. Departamento de Ingeniería Química, Universidad de Sevilla, 41012 Sevilla, Spain;2. Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway;1. Department of Neurology, National Neuroscience Institute, Tan Tock Seng Hospital, Singapore;2. Department of Neurology, National Neuroscience Institute, Singapore General Hospital, Singapore;3. Neuroscience and Behavioural Disorders Program, Duke-NUS Medical School, Singapore;1. Servicio de Reumatología, Instituto de Investigación Germans Trias i Pujol, Badalona, Barcelona, España;2. Department of General Medicine, Borders General Hospital, Melrose, Scotland, Reino Unido;3. Servicio de Medicina Interna, Hospital Universitario Central de Asturias, Oviedo, Asturias, España;1. Department of Biology, University of Maryland, United States;2. Department of Pharmacology and Toxicology, University of Louisville, United States;3. Department of Biochemistry & Molecular Biology, Medical University of South Carolina, United States;4. Department of Chemistry and Biochemistry, City University of New York, Queens College, United States |
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| Abstract: | Collagenase from normal human skin fibroblasts was found to catalyze the hydrolysis of esters and thio esters. This observation led to the development of a rapid, sensitive, continuous spectrophotometric assay for vertebrate collagenase using the thio peptolide Ac-ProLeuGly-S-LeuLeuGly-OC2H5 as substrate in the presence of 4,4'-dithiodipyridine or Ellman's Reagent. A Km of 0.004 M and a kcat of 370,000 h-1 were determined for the thio peptolide-enzyme reaction. The method is able to detect collagenase at concentrations as low as 2 ng/ml. |
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