Proteomics application of SAT - activity-based protein profiling


Measurement of the activity of proteolytic enzymes and nucleases

Activity of hydrolytic enzymes like proteases or nucleases, using fluorescent substrates immobilized on microbeads, appeared to be the candidates of choice for such applications.

The microbead system proved to be valuable for the following purposes.
  1. protease assays using (biotin and FITC-labeled) protein or peptide protease substrates. Using casein-FITC/biotin, we could detect as little as a single molecule of proteinase K.
  2. nuclease assays/RFLP studies using PCR products prepared by biotinylated forward and fluorescent reverse primers and a restriction enzyme that cleaves the DNA molecule at a particular site
The spectrum of potential applications include detection of serum proteinases and nucleases in diseases involving massive tissue destruction: following heart-attack, acute or chronic necrotic processes, including malignancies, proteolytic enzyme profiling in tumor biopsies, detection of metalloproteinases in synovial fluids derived from patients afflicted with autoimmune diseases, and measurement of the activity of certain key proteolytic enzymes involved in hemostasis. The technology can also be used for measurement of HIV protease, both for HIV detection purposes and for experimental applications in projects aimed at developing HIV protease inhibitors.


Proteinase K titration with casein-biotin-FITC.
Sensitivity of assay: 1 proteinase K molecule /sample.
 Fluctuation analysis: digetsion results of parallel aliquots containing, on the average, 1 molecule of proteinase K