By Arnold Joseph Toynbee, Royal Institute of International Affairs
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WISEMAN, MICHAEL GREEN, and JuDD BERMAN Introduction The optimization of substrate design is an initial step toward the development of robust enzyme assays. In addition, the information gained from the optimization procedure can serve as a basis for inhibitor design. We previously reported a method which allows rapid optimization of substrates by screening pools of specific peptide mixtures. 1 Optimal substrates can be directly identified in each pool by using a combined high-performance liquid chromatography/mass spectrometry (HPLC/MS) technique.
E. Danley, L. G. , and G. C. Andrews, FEBS Lett. 262, 119 (1990). 45 N. M. Green, Biochem. f. 90, 564 (1964). 46 A. J. Barrett, C. G. Knight, M. A. Brown, and U. Tisljar, Biochem. f. 260, 259 (1989). S. Stack and R. D. Gray, f. Bioi. Chern. 264, 4277 (1989). 42  FLUORIMETRIC ASSAYS OF PROTEOLYTIC ENZYMES 29 valuable tools in studies of the matrix metalloproteinases, 48 A 9 there are disadvantages to the use of tryptophan as a fluorophore. 2. Tryptophan is also abundant in proteins, so that assays made with crude enzyme preparations, or with protein inhibitors, will be less sensitive owing to a high fluorescence background.
Nixon, K. M. K. Bottomley, M. J. Broadhurst, P. A. Brown, W. H. Johnson, G. Lawton, J. Marley, A. D. Sedgwick, and S. E. Wilkinson, Int. J. Tissue React. 13, 237 (1991). 19 H. J. Andrews, T. A. Plumpton, G. P. Harper, and T. E. Cawston, Agents Actions 37, 147 (1992). 20 C. B. Caputo, L. A. Sygowski, D. J. Wolanin, S. P. Patton, R. G. Caccese, A. Shaw, R. A. Roberts, and G. DiPasquale, J. Pharmacal. Exp. Ther. 240, 460 (1987). 21 D. J. Buttle, C. J. Handley, M. Z. Ilic, J. Saklatvala, M. Murata, and A.