Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

✍ Scribed by George C. Saunders; Zita Svitra; Adelmo Martinez

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 712 KB
• Volume: 126
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90117-8

✦ Synopsis

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quart&ted calorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 rig/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2'-azino-Di-(3-ethyl-benzthiazoline-6-sulfonate)-HrOz.

For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.