Steady-state fluorescence quenching

M. F. Blackwell, K. Gounaris, S. J. Zara, and J. Barber, A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies, Biophys. J. 51, 735-744 (1987). 

While no spectroscopic evidence of a ground-state complex between anthracene and carbon tetrachloride, naphthalene or 1,2-benzanthracene and carbon tetrabromide has been found, Nemzek and Ware [7] were unable to explain their steady-state fluorescence quenching measurements with the parameters deduced from the determination of the time-dependent rate coefficients unless a ground-state complex was present. This cannot be regarded as a satisfactory and consistent analysis because the time-dependent rate coefficient would be modified by the presence of the initial distribution of quencher and fluorophor in the ground state. 

Several singlet-excited cations have been shown to imdergo photoinduced electron transfer from aromatic donors. Fluorescence from the 9-phenylxanthyl, xanthyl, thioxanthyl, and 9-phenylthioxanthyl cations is quenched in the presence of aromatic compounds [9,10,15], Steady-state fluorescence quenching experiments gave bimolecular excited state rate constants for quenching of the cations by the aromatic donors (Table 7). The quenching rate constants increase... 

Figure 6. Steady state fluorescence quenching data fluorescence intensity in the absence (/o) and presence (/) of quenchers, z = mean occupation number of quencher molecules per micelle (proportional to analytical quencher concentration). Curve a k = Icq, Eq. 43 curve b k = Icq, Eq. 41 curve c yi = 1 + z/2 (Stem-Volmer reference line).

Figure 7. Steady state fluorescence quenching as a function of the relative magnitudes of the rate constants for quenching, k (or k) and for radiative transition, k. Solid lines Eq. 43, broken lines Eq. 41. Curve a k = 100 k ik = 100 Ao). Curve f> k = 10 Ao (A = 10 Ao). Curve c A = Ao (k = Aq).

Matyus L, Szoflosi J, Jenei A (2006) Steady-state fluorescence quenching applications for studying protein structure and dynamics. J Photochem Pholobiol B Biol 83 223-236... 

By using the information obtained when the emission of the probe molecule is quenched, i.e. either by eximer formation of the probe itself or by addition of a molecule that quenches the exited state of the probe, it is possible to further characterize the system in terms of micellar shape. This is a technique frequently used in a wide variety of systems, including binary water-surfactant or polymer-surfactant systems, or microemulsions. Two methods are worth extra attention, namely the steady-state fluorescence quenching (SSFQ) and the time-resolved fluorescence quenching (TRFQ) methods. For more comprehensive texts describing these techniques, the reader is referred to refs (21) and (22). 

Nonradiative reiaxation and quenching processes wiii aiso affect the quantum yieid of fluorescence, ( )p = /cj /(/cj + Rsiative measurements of fluorescence quantum yieid at different quencher concentrations are easiiy made in steady state measurements absoiute measurements (to detemrine /cpjj ) are most easiiy obtained by comparisons of steady state fluorescence intensity with a fluorescence standard. The usefuiness of this situation for transient studies... 

The importance of comparing time-dependent and steady-state fluorescence measurements is well illustrated by the difficulty of resolving purely static from purely dynamic quenching. In either case, the basic relationship between the steady-state fluorescence intensity and quencher concentration is the same. The Stem-Volmer relationship for static quenching due to formation of an intermolecular complex is i... 

By comparing time-resolved and steady-state fluorescence parameters, Ross et alm> have shown that in oxytocin, a lactation and uterine contraction hormone in mammals, the internal disulfide bridge quenches the fluorescence of the single tyrosine by a static mechanism. The quenching complex was attributed to an interaction between one C — tyrosine rotamer and the disulfide bond. Swadesh et al.(()<>> have studied the dithiothreitol quenching of the six tyrosine residues in ribonuclease A. They carefully examined the steady-state criteria that are useful for distinguishing pure static from pure dynamic quenching by consideration of the Smoluchowski equation(70) for the diffusion-controlled bimolecular rate constant k0,... 

In systems where only dynamic quenching occurs, then steady-state fluorescence intensities can be measured instead of lifetimes/101 103-,07) In experiments where comparisons are being made (i.e., for a comparison of different experimental conditions or types of membrane), it is important that the lifetime of the fluorophore (r0) is not affected by the experimental conditions. Fluorescence intensities can be obtained much more rapidly and without specialized instrumentation. Blatt and Sawyer(101) have employed a relationship essentially the same as Eq. (5.20) in this way. They have pointed out that since the quenching mechanism is collisional, the partition coefficient that is derived is a partition coefficient of the quencher into the immediate environment of the fluorophore and is therefore a local Kp. It is therefore possible to investigate the partition coefficient gradient across the lipid bilayer by using a series of probes, such as the anthroylstearates,(108) located at different depths. In their method, Eq. (5.20) has the form... 

After extraction, the fluorescent indicator was in the unbound state and gave input to the radiative relaxation. Therefore, the fluorescence lifetime increased and, consequently, the intensity as well. After MIP contacting with the analyte, the non-radiative processes were again efficient compared to the radiative processes and, subsequently, fluorescence was quenched. With steady-state fluorescence spectroscopy the cross-reactivity test towards structurally similar biomolecules was performed that yielded selectivity factors for guanosine, cAMP and cCMP of 1.5, 2.5 and 5.1, respectively. 

Steady-state fluorescence spectra recorded after the addition of the NDI or PM I acceptor to the bisporphyrin tweezer ( rrl = 660 nm), demonstrated substantial quenching (75%) with increasing quantities of the NDI or PM I acceptors. Time-resolved emission spectra recorded in toluene for the complex 26 were biexponential containing a dominant short-lived CS components (80 ps, -95%) attributed to photoinduced ET from donor porphyrin to NDI, and a minor long-lived component (Ins, 5%). The lifetime of the dominant short-lived CS state is increased two- to threefold relative to covalently linked systems under similar conditions of solvent, donor-acceptor distance and thermodynamics [37]. Charge recombination rates from 1.4 to 3.8 x 1()9s 1 were observed, depending on whether the NDI or PM I acceptor was bound within the cavity. 

Staab and coworkers have prepared stacked Q-P-Q triad 28 [80-83]. An X-ray structure determination of the molecule shows that the quinones are situated directly above and below the plane of the porphyrin, with their planes parallel to the porphyrin plane and 3.4 A from it. Steady state fluorescence measurements demonstrate strong quenching of the porphyrin first excited singlet state, which in turn suggests rapid electron transfer. Additional data were obtained from fluorescence lifetime measurements [83]. In dichloromethane, for instance, the lifetime of an analog of 28 in which the quinones were replaced with redox inactive dimethoxyphenyl substituents was 9.0 ns. In 28, this lifetime was reduced to 2 ) ps. 

Sanders and coworkers have prepared an interesting molecular assembly 32 in which the central porphyrin moiety is held in a sandwich conformation between the two pyromellitimide acceptors by coordination of its pyridyl groups with the zinc ions of the two peripheral porphyrin moieties [86]. Steady state fluorescence studies revealed that the fluorescence of the central porphyrin was quenched by at least 90% relative to the unbound material. This was interpreted in terms of electron transfer to the pyromellitimide moieties. Of course, energy transfer from the metallated porphyrins to the central free base porphyrin might also be expected in this molecule. 

The presence of H2P and ZnP in the triads 15a-c is reflected by their strong emission, which dominates large parts of the steady-state fluorescence spectra. However, in comparison to the H2P/ZnP references the porphyrin centered fluorescence is appreciably quenched in all H2P/ZnP-oPPE -C60. Interestingly, this... 

Turning to steady-state fluorescence studies, appreciable quenching of Cso emission at 710 nm was only discernable for the monomer 17a, i.e. 

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