Slow Fluorescence Component Research Articles

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Photo-dynamics of roseoflavin and riboflavin in aqueous and organic solvents

Roseoflavin (8-dimethylamino-8-demethyl- d-riboflavin) and riboflavin in aqueous and organic solvents are studied by optical absorption spectroscopy, fluorescence spectroscopy, and fluorescence decay kinetics. Solvent polarity dependent absorption shifts are observed. The fluorescence quantum yields are solvent dependent. For roseoflavin the fluorescence decay shows a bi-exponential dependence (ps to sub-ps time constant, and 100 ps to a few ns time constant). The roseoflavin photo-dynamics is explained in terms of fast intra-molecular charge transfer (diabatic electron transfer) from the dimethylamino electron donor group to the pteridin carbonyl electron acceptor followed by intra-molecular charge recombination. The fast fluorescence component is due to direct locally-excited-state emission, and the slow fluorescence component is due to delayed locally-excited-state emission and charge transfer state emission. The fluorescence decay of riboflavin is mono-exponential. The S 1-state potential energy surface is determined by vibronic relaxation and solvation dynamics due to excited-state dipole moment changes (adiabatic optical electron transfer).

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Time-resolved experiments on external microwave field action in gaseous oxalylfluoride excited to the 00 0—band of the μ1Au state

Time-resolved measurements of the microwave field effect using optically detected EPR (ODEPR) have demonstrated that the amplitude and lifetime of the slow component of fluorescence are additionally reduced by an external microwave field, at a microwave frequency of 9400 MHz, a constant magnetic field of 0.3295 T and an oxalylfluoride pressure of 30 mTorr. This is accompanied by an increase in the fast component amplitude, at a constant decay rate of (2.36 × 0.19) 107 s−1. The fluorescence intensity was found to decrease, and phosphorescence intensity to increase, with subsequent saturation at higher microwave intensities. The experimental data are interpreted using the indirect mechanism theory in the limit of low-level density.

Electric Field Effects on Steady State and Time Resolved Fluorescence from Photosynthetic Reaction Centers

Abstract A promising approach to identifying the primary electron acceptor and thereby the mechanism of primary charge separation in photosynthetic reaction centers (RCs) is the orientational determination of the dipole moment of the primary radical pair. This can be achieved by studying the angular dependence of the primary charge separation rate in an external electric field as reflected in the Dichroic Excitation spectrum of the electric Field modulated Yield of the prompt fluorescence (DELFY). For RCs from Rb.sphaeroides R-26 steady state, low temperature DELFY experiments point (within an angle of 5°) to the orientation of the dipole moment of P+H− A. Time resolved fluorescence measurements revealed that the major contribution to the steady state fluorescence quantum yield and to the electric field effect thereon originates from a slow component with a lifetime of ≃ 300 ps at 80K. This fluorescence component is by two orders of magnitude slower than the primary charge separation rate measured in absorption and might originate from a small (≃3%) subset of RCs characterized by slow, unistep charge separation. Recent transient absorption measurements in electric fields revealed a significant reduction of the quantum yield of P+H− A formation within 30ps demanding a fast loss channel. In order to achieve dynamic competition with fast charge separation in the majority of RCs a more efficient loss channel than internal conversion is required, such as fast parking or trapping of excitation energy. From such a field modulated trapping state with the ability to fluoresce one would expect the observed loss of P+H− A quantum yield accompanied by a slow fluorescence component, which should be stronger than the measured one, however. This model would imply direct charge separation from 1P* to P+H− A in one step in the majority of RCs. Electric field induced charge separation to the B-branch forming P+B− B would better comply with the data, in case its energy is high enough to produce a quadratic field dependence of the delayed recombination fluorescence.

Dual Fluorescence of Pyrazine Cooled in a Supersonic Free Jet

Abstract The fluorescence excitation spectrum of the lowest excited 1B3u(n π*) state of pyrazine cooled in a pulsed supersonic free jet was observed. The fluorescence decay of the isolated molecule was also measured in the free jet. The rotationally cooled pyrazine in the collision free condition exhibits extremely large fluorescence quantum yield and dominant slow fluorescence component for low lying vibronic levels. The ratio of the pre-exponential factors of the dual fluorescence decay was examined in terms of the available theory of singlet-triplet mixed state model. It was found that the number of coupled triplet levels obtained from the observed ratio is inconsistent with the calculated excess energy dependence of vibronic density in the triplet state. Remarkable rotational level dependence of the ratio of the pre-exponential factors was observed for the electronic origin, while such a rotational dependence was not found for the lifetime of the slow fluorescence.

Rotational effects on the quantum yield and lifetime of the slow component of fluorescence from s-triazine

The fluorescence quantum yield and lifetime of the slow component of fluorescence obtained along the rotational contour of the 6 1 0 and 6 2 0 absorption bands of s-triazine at low pressure show a marked variation. For each band, the quantum-yield spectrum shows a sharp peak at the Q-branch edge, the lifetime spectrum exhibits a valley at the same position.

Effects of Linolenic Acid on the Spectral Properties and Picosecond Fluorescence of Pea Chloroplasts

Abstract Linolenic acid induces changes in the absorption spectrum and in the picosecond fluorescence of pea chloroplasts. The effects of linolenic acid are dependent on concentration and time. Linolenic acid increases the fluorescence life time of chloroplasts at room temperature. The contribution of the slow fluorescence component relative to the fast component is increased almost 10 fold in the presence of 0.5 mᴍ linolenic acid. The synergistic action of digitonin and linolenic acid increases the ratio of “closed” to “open” traps in the photosynthetic units. Upon addition of 0.5 mᴍ linolenic acid there are increases in absorbance at 676 and 436 nm, and decreases in absorbance at 705 and 496 nm. Some of the spectral changes have a biphasic character, they reach a maxi um um after about 30 min then start to reverse. Based on the spectral changes at 496 and 705 nm it appears that linolenic acid has at least two effects, i.e. it modifies the thylakoid membrane and secondarily decreases the concentration of P700, respectively.

Effects of methyl substitution on electronic relaxation processes of pyrimidine vapor

The pressure and excitation-energy dependence of the fluorescence quantum yields of pyrimidine, 5- and 4-methylpyrimidines and 4,6-dimethylpyrimidine in the vapor phase has been investigated in the pressure range 10 −3–10 2 torr. The results indicate that not only pyrimidine but also its methyl derivatives give both fast and slow fluorescence emissions at low pressure, in conformity with the intermediate case of electronic relaxation. The total fluorescence quantum yields for the 0-0 transition, extrapolated to zero pressure, are 0. 17, 0.0079, 0.0057 and 0.0010 for pyrimidine, 5- and 4-methylpyrimidines and 4,6-dimethylpyrimidine, respectively. The methyl substitution significantly reduces the yield of the slow fluorescence component in essentially the same manner as the increase of excitation energy in the unsubstituted pyrimidine. The decrease of the slow-fluorescence yield resulting from the methyl substitution or the excitation-energy increase is interpreted as due to the increase of the number of triplet vibronic It is shown that there is a fairly good correlation between the number of the effectively coupled triplet levels and the vibrational state density in t

Fast and slow fluorescence emissions from pyrimidine vapor

The pressure and excitation-energy dependence of the fluorescence quantum yield and lifetime of pyrimidine vapor has been investigated in the pressure range 10 −3-10 torr. The results indicate that in conformity to the intermediate case the fluorescence of the isolated pyrimidine molecule consists of fast and slow components with lifetimes of the order of 1 ns and 10 μs, respectively. The total fluorescence quantum yield amounts to as high as 0.045. The yield of the slow fluorescence component decreases significantly with increasing excitation energy; this observation is interpreted as being due mainly to the lengthening of the radiative lifetime of that component.

Slow, fast and dual fluorescence in glyoxal vapor

We studied the fluorescence decay behavior of glyoxal at low pressure at various excitation energies. At low excitation only a slow fluorescence component was found, at higher energies we observed a fast fluorescence component in addition to the slow one. It was concluded that strong vibronic singlet-triplet mixing occurs only at higher excitations due to the increasing triplet level density. Numbers parametrizing this coupling were obtained. We also point out in which excitation region oscillatory decay behavior should be expected. The lifetime of the slow fluorescence appears to strongly decrease with increasing vibrational excess energy. From this we primarily conclude that the zero order triplet decay rate is strongly dependent on triplet vibrational excess energy.