Send to printer »

Insight & Intelligence™ : Nov 5, 2012

ATP Gets Smart/Absorbing BLA Assay

Researchers propose a fluorescent probe for adenosine triphosphate assay and describe a synthesis of a chromogenic cephalosporin for β-lactamase assay.
  • Doug Auld, Ph.D.

Bioluminescent methods exist to detect ATP that require coupling to firefly luciferase, an ATP-dependent enzyme. The assay described in the first article* uses a molecular beacon approach and an ATP-dependent DNA ligase. The ligase reaction proceeds through three steps: adenylation of an active site residue of the ligase to yield an enzyme-bound AMP with release of pyrophosphate, transfer of the AMP to the 5′-end of an oligonucleotide to yield a pyrophosphate bond, and formation of the 5′–3′ phosphodiester bond in the DNA.

In the assay, two oligonucleotides are hybridized to a “smart probe,” which is a DNA hairpin containing both a fluorophore and quencher at its ends (see Figure 1). Hybridization of the two oligonucleotides does not result in hairpin disruption, but upon ligation the resulting longer oligonucleotide opens the hairpin and relieves the quenching of the fluorophore (see Figure 1). The detection limit was found to be 0.5 nM (3 SD over background). This sensitivity is approximately one order of magnitude higher than bioluminescent approaches.

The second article by Yu et al.** describes the synthesis of a new chromogenic substrate for β-lactamase (bla). The bla enzyme is a commonly used reporter in cell-based assays in which a fluorescence-resonance-energy-transfer (FRET) substrate is used to measure activity.

The FRET substrate CCF2, consisting of a cephalosporin core linking 7-hydroxycoumarin to a fluorescein moiety, results in blue fluorescence when the substrate is cleaved by bla and green fluorescence when uncleaved, therefore a ratiometric assay is enabled using the blue/green fluorescence value. However, CCF2 is expensive as is Nitrocefin (the most commonly used absorbance-based substrate) and therefore the authors sought to synthesize improved absorbance-based substrates.

The authors describe a two-step synthesis to synthesize a chromogenic cephalosporin-[3-(4-nitrostyryl)-7-(2-phenylacetamido)-ceph-3-em-4-carboxylicacid (Chromacef). Cleavage of the β-lactam ring by bla results in a bathochromic shift of 64 nm (λmax from 378 nm to 442 nm; see Figure 2). The KM value for Chromacef using VIM-2 metallo-β-lactamase was 30 ± 7 μM and for TEM-1 it was 51 ± 15 μM, which are values similar to that obtained for Nitrocefin. The kcat values were improved compared to Nitrocefin.

Chromacef was shown to be cleaved by a variety of β-lactamases and should be a useful substrate to develop bla assays as well as serve as an orthogonal substrate.

*Abstract from Analytical Biochemistry 2012, Vol. 429: 8–10

A novel fluorescent probe for adenosine triphosphate (ATP) assay based on DNA ligation is proposed in this article. This approach uses a novel smart probe, T4 DNA ligase, and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction and the ligation product restores the fluorescence of the smart probe. This method is very sensitive with a 0.5-nM limit of detection. Compared with current assay methods, the strategy is simpler, cheaper, and 40 times more sensitive.

**Abstract from Analytical Biochemistry 2012, Vol. 428: 96–98

Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λmax undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δ442 nm of 14,500 cm−1·M−1. This compound was shown to be a substrate for a variety of β-lactamases.