Determining Protease Substrate Selectivity and Inhibition by Label-Free Supramolecular Tandem Enzyme Assays

Abstract

An analytical method has been developed for the continuous monitoring of protease activity on unlabelled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit [7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively recognizing N-terminal phenylalanine residues as they are produced during the enzymatic cleavage of enkephalintype peptides by the metalloendopeptidase thermolysin. The substrate peptides (e.g., ThrGly-Ala-Phe-Met-NH2) bind to CB7 with moderately high affinity (K ca. 10 4 M -1 ), while their cleavage products (e.g., Phe-Met-NH2) bind very tightly (K > 10 6 M -1 ). AO signals the reaction upon its selective displacement from the macrocycle by the high affinity product of proteolysis. The resulting supramolecular tandem enzyme assay effectively measures the kinetics of thermolysin, including the accurate determination of sequence specificity (Ser and Gly instead of Ala), stereospecificity (DAla instead of LAla), endo-versus exopeptidase activity (indicated by differences in absolute fluorescence response), and sensitivity to terminal charges (-CONH2 versus -COOH). The capability of the tandem assay to measure protease inhibition constants was demonstrated on phosphoramidon as a known inhibitor to afford an inhibition constant of (17.8 ± 0.4) nM. This robust and label-free approach to the study of protease activity and inhibition should be transferable to other endo-and exopeptidases that afford products with N-terminal aromatic amino acids.2

In vitro inhibition of lysine decarboxylase activity by organophosphate esters

“…The fluorescence intensity at 525 nm relative to the one without the CB7 addition was plotted as a function of the total CB7 concentration. Using the modified Benesi-Hildebrand equation [41], the association constant of AO with CB7 was calculated to be 1.50 Â 10 5 M À1 , which is agreeable with the reported value (2.90 Â 10 5 M À1 ) [42]. Based on the fluorescence titration curve, the concentrations of CB7 and AO were chosen to be 5 mM and 0.5 mM respectively.…”

“…But unfortunately, LDC consumption in these systems is quite high (40.0 mg mL À1 ). Recently, the same group employed the CB7/AO pair for the monitoring of protease activity and inhibition [42]. We therefore tested the possibility of using this system to reduce the enzyme consumption in LDC activity assay and subsequently used it to screen a large number of inhibitors for LDC.…”

Supramolecular Approach to Enzyme Sensing on Paper Discs Using Lanthanide Photoluminescence

“…The most commonly employed enzyme assays are solution based and the detection is done either through colorimetry [29][30][31][32][33] or fluorimetry. 34,35 Although the fluorescence based assays provide a higher intrinsic sensitivity, the autofluorescence from biological samples interferes with the measurements and this is a drawback for the detection of bioanalytes. In this context, a paper based sensing platform to detect enzyme activity through a delayed luminescence read-out would be more convenient.…”

A novel synthesis of nitroform by the nitrolysis of cucurbituril

“…When reacted with unsaturated com-13 pounds, NF afforded multi-nitroalkane derivatives, therefore it 14 can be used for the synthesis of a system explosive containing 15 nitroform group, such as 1-(2,2,2-trinitroethylamino)-2-nitrogua- 16 nidines [4] and 1,1,1,3-tetranitro-3-azabutane [5]. Due to the 17 acidic hydrogen atom, nitroform can generate a lot of nitroformate 18 salt derivatives, for example hydrazinium nitroformate (HNF).…”

“…Over the past decade, the particular properties of 39 cucurbiturils, including the binding of positively charged molecules 40 at the two carbonyl portals and of neutral molecules in the 41 hydrophobic cavity, are the focus of attention in supramolecular 42 chemistry. They have been widely used in many fields, such as drug 43 delivery [12], molecular machines [13], supramolecular polymers 44 [14], sensing ensembles [15], and biomimetic systems [16]. 45 Thought of the carbonyl groups of CB [6] substituted by nitro 46 groups, a special class of high energy density compound may be 47 devised (Fig.…”