Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes

Abstract

We introduce a new economic, convenient and general assay principle based on the reversible interaction of water-soluble macrocycles and fluorescent dyes. We show that amino acid decarboxylase activity can be continuously monitored by measuring changes in fluorescence, which result from the competition of the enzymatic product and the dye for forming a complex with a cucurbituril or calixarene macrocycle. The new assay provides a complementary method to the use of antibodies, radioactive markers and labeled substrates.

In vitro inhibition of lysine decarboxylase activity by organophosphate esters

“…The relative fluorescence intensity at 510 nm was plotted as a function of the competitor concentration. The association constants of CB7 with lysine and cadaverine were estimated to be 1.01 Â 10 3 M À1 and 4.94 Â 10 6 M À1 , which were in good accordance with previous reports, 8.70 Â 10 2 M À1 for lysine and 1.40 Â 10 7 M À1 for cadaverine [43]. Obviously, cadaverine has much higher affinity with CB7 than lysine.…”

“…Because both the substrate and product of LDC are optically inert, it is difficult to develop colorimetric or fluorescence assays based on conventional methods. In 2007, Henning et al described a novel concept for the determination of enzyme activity of amino acid decarboxylases, using macrocyclic receptors and fluorescent dyes such as CB7/Dapoxyl and CX4/DBO [43]. This competitive displacement method is simple and convenient.…”

“…The optimal concentrations of LDC and lysine were determined to be 8.0 mg mL À1 and 50 mM. The limit of detection for LDC is 0.25 mg mL À1 (signal-to-noise ratio of 3), which is much lower than the values in the CB7/Dapoxyl and CX4/DBO systems [43]. After addition of 8.0 mg mL À1 LDC and 50 mM lysine into a solution of 0.5 mM AO and 5 mM CB7, the fluorescence peak redshifted gradually to 525 nm, and the intensity decreased with time until it reached the background (Fig.…”

Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

“…Therefore various label-free methods for enzyme activity measurements have been developed, based on different techniques, such as liquid chromatography-mass spectrometry (LC-MS) [3], capillary electrophoresis [4,5], a method based on macrocycle-fluorescent dye complex formation [6], and methods based on heat measurements with isothermal titration calorimetry (ITC) [7]. Recently, a method for timeresolved initial rate measurements using isothermal titration calorimetry (ITC) has been developed as a generic assay for the activity of enzymes.…”

A supramolecular keypad lock

“…22,23 Cucurbiturils have drawn much attention for their very high binding constants of cationic guests (up to 10 17 M À1 ) 24 and their supramolecular application potential with a strong focus on biological and pharmacological contexts, and analytical problems is beginning to reveal. [25][26][27][28][29][30][31][32][33][34][35] In the present work we take advantage of the specific complexation properties of the CB8 macrocycle. Unlike the smaller homologues CB6 and CB7, that commonly offer space for only one guest molecule, the larger CB8 often accommodates two guests and the resulting complexes may feature new emission properties (e.g., excimer fluorescence) or lead to fluorescence self-quenching.…”