| Assay Method Information | |
| | Enzyme Assay (Cruzain) |
| Description: | All enzyme assays were performed at 25° C. Initial rates of the peptidolytic reaction catalyzed by cruzain were measured by monitoring the fluorescence generated by cleavage of the dipeptide-AMC bond. Assays were conducted in 96-well plates (Greiner; flat-bottom, clear black plates) in a total volume of 250 μL, containing either 50 mM MES (pH 7.5), 50 mM TAPSO, 100 mM DEA, 1 mM CHAPS, 1 mM Na2EDTA, 5 mM DTT, and 10% DMSO (v/v) or 50 mM sodium acetate (pH 5.5), 50 mM MES, 100 mM TEA, 1 mM CHAPS, 1 mM Na2EDTA, 5 mM DTT, and 10% DMSO (v/v). Substrates were dissolved in 100% DMSO and were then diluted 10-fold such that when added to reaction mixtures, final DMSO concentration was 10% (v/v). Reactions were initiated with the addition of 1-10 μL of cruzain (final concentrations: 0.1-3.0 nM (preincubation studies)). Fluorescence was measured on either a SpectraMax M5 (Molecular Devices) or a Synergy HTX (BioTek, Winooski, VT) microplate reader (λex=360 nm, λem=460 nm). Initial rates were determined from continuous kinetic time courses and calculated from the earliest time points, typically at less than 10 min.Compounds were evaluated as inhibitors or inactivators of cruzain in two ways: (1) enzyme was added to reaction mixtures containing the substrate (typically, 10 μM Cbz-Phe-Arg-AMC) and inhibitor, and reaction time courses were measured for 0-40 min. In addition to other methods, the effects of all inhibitors on reaction rates were determined at t=0-200 s (vi) and at longer incubation times (t>1000 s; vs), to ascertain the respective inhibition constants Ki and Ki*. (2) Enzyme and compound were preincubated over extended periods of time, and then aliquots were removed and diluted 50-fold to 100-fold into reaction mixtures containing 10 μM Cbz-Phe-Arg-AMC, followed by the assessment of the resulting time courses. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |