Fret Substrates

Fret Substrates offered by Bachem

Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from an excited fluorophore (or donor) to a suitable quencher (or acceptor) molecule. FRET is used in a variety of applications including the measurement of protease activity with substrates, in which the fluorophore is separated from the quencher by a short peptide sequence containing the enzyme cleavage site. Proteolysis of the peptide results in fluorescence as the fluorophore and quencher are separated. In this brochure we present a range of highly sensitive FRET protease substrates for a variety of enzymes.

Introduction

Fluorophores are substances which, like chromophores, absorb light in the UV or visible range. In contrast to chromophores they re-emit part of the light as radiation. This process is called fluorescence and is illustrated by the Jablonski energy level diagram (Fig 1). Absorption of light (hνA) causes an electron to be promoted from its electronic ground state (designated as S0) to an excited state (usually S1).

Every energy state has several vibrational energy levels 0, 1, 2 etc. During the lifetime of the excited state, i.e. the time elapsed between excitation of the molecule and emission of the photon (usually between 1-10 ns), part of the energy is lost by internal vibration. As a result, the wavelength of the emitted light (hνF) is always longer than that of the exciting light.

This phenomenon is called the Stokes shift and allows the detection of emission against a background of light derived from excitation. Usually, the fluorescence excitation spectrum of a fluorophore in a diluted solution is identical to its absorption spectrum and under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength.

In a diluted solution, fluorescence intensity is linearly proportional to several parameters as deduced from Lambert-Beer’s law. These are the molar absorption coefficient, the path length, the intensity of the incident light, and the quantum yield which is the ratio of the number of emitted to the total number of absorbed photons. Fluorescence detection is dependent on the sensitivity of the instrument and is therefore measured in arbitrary units.

Higher concentrations of the fluorophore (> 0.1 absorption units) lead to deviations from the linearity due to loss of excitation intensity across the cuvette path length as the excitation light is absorbed by the fluorophore. This phenomenon is known as the inner filter effect. Other effects which influence fluorescence measurements are related to intrinsic or background fluorescence originating from sample preparations and buffer contaminants, respectively. To minimize fluorescence derived from contaminants, it is recommended to use materials of maximum purity.

Fluorescence spectra may also be dependent on the solvent. With some fluorophores, such as 2-acetylanthracene or tryptophan, a spectral shift to longer wavelengths (bathochromic shift or red shift) is observed in more polar solvents. The fluorescence spectra of fluorophores containing acidic or basic substituents (e.g. AMC) can depend on the pH of the solution.

Fig.1. Energy Level Diagram

Fig. 2. Fluorescence Resonance Energy Transfer (FRET)

Table 1. Fluorophores

* the values listed are as reported in the cited literature.

Table 2. Donor/Acceptor Pairs