Kinetics of rearrangement of solvation sheath of an excited molecule of the fluorescent probe 4"-dimethylaminochalcone

Processes accompanying the quenching of the fluorescent probe 4"-dimethylaminochalcone by hydroxyl groups of the proton-donor solvent 1-butanol have been studied. The kinetics of the deactivation of the excited state of 4"-dimethylaminochalcone has been monitored from the transition absorption spectra at a time resolution of 50 fs and fluorescence decay at a time resolution of 30 ps. The data obtained allow thinking that the next picture occurs in 1-butanol. At first stage, the 4"-dimethylaminochalcone molecule in its ground state forms a hydrogen bond with an alcohol molecule. At the second stage, the absorption of light quantum and corresponding rise of the dipole moment of 4"-dimethylaminochalcone take place, the initially existing hydrogen bond is retained. The third stage consists in the rearrangement of the 4"-dimethylaminochalcone solvation shell formed by alcohol dipole molecules due to an increase of the dipole of moment 4"-dimethylaminochalcone; this rearrangement takes an energy of about 24 kJ/mol, the arrangement time constant is close to 40 ps; the initial hydrogen bond is retained. The fourth stage involves processes that lead to fluorescence quenching; their time constant is about 200 ps. Taking into account that the quenching is a much slower process than the relaxation of the solvation shell, it was supposed that the quenching is not a direct consequence of the solvation shell relaxation or the existence of the hydrogen bond formed prior to excitation. Then the fluorescence quenching of 4"-dimethylaminochalcone can be accomplished through some other processes that are observed in other fluorescent molecules: (a) rearrangement of the initial hydrogen bond from a conformation that cannot quench the fluorescence of 4"-dimethylaminochalcone to a more "effective" conformation, (b) charge transfer between the excited of molecule 4"-dimethylaminochalcone and alcohol, or (c) solvent-induced twist of the 4"-dimethylaminochalcone amino group (its withdrawal from the molecule plane) by the action of the solvent.     

The lipophilic fluorescent probe 4-dimethylaminochalcone: factors responsible for the fluorescence yield

Factors responsible for fluorescence quenching of the lipophylic fluorescent probe 4-dimethylaminochalcone in nonpolar and polar media were studied. The femtosecond dynamics of 4-dimethylaminochalcone excited state was measured using the absorption method of "excitation probing". In nonpolar hexane where the fluorescence quantum yield is very low (0.001), all excited 4-dimethylaminochalcone molecules go to the triplet state with a rate constant of 2.10(11) s(-1). At the same time, the radiation rate constant is 1000 times lower; therefore, such a fast transition to triplet is the major cause of the very small fluorescence yield. In polar acetone, the fluorescence yield is 220 times higher than in hexane. In acetone, no transitions to triplet state were detected. At the same time, a radiationless conversion to the ground state took place with a rate constant of 10(9) s(-1), which decreased the fluorescence yield to 0.22. The activation energy of the quenching processes is polarity dependent and decreases from 6 in nonpolar to 3 kcal/mol in polar media. The yield of 4-dimethylaminochalcone fluorescence varies hundreds times in media of different polarity but is a linear function of the Lippert's polarity parameter f(epsilon,n) where epsilon is the dielectric constant at low frequencies. It is supposed that polar media stabilize the "flat" conformation of the 4-dimethylaminochalcone molecule prior to its excitation, and this conformation is optimal for fluorescence. In this case, stabilization is determined only by medium polarity.     

Molecular mobility of a fluorescent probe in binding sites of an albumin molecule

The molecular mobility of the fluorescent probe, N-(carboxymethyl)imide of 4-(dimethylamino)naphthalic acid (K-35), in three types of binding site on a human serum albumin (HSA) molecule has been studied. Study of the time-resolved decay of K-35 polarized fluorescence in HSA has shown that probe molecules bound to different sites have different fluorescence decay times, which poses problems in interpreting the polarization curves. However, it has been found that, in the case of rather slow thermal rotation of the probe, the decay of the vertical and the horizontal components of polarized fluorescence can each be approximated with three exponentials corresponding to three types of binding site. The mobility of the probe in different sites was estimated. The mobility was different but in all cases hindered by tens of times relative to the rotation of K-35 in water. The slowest motion occurred in the sites of the first type localized in the region of the well known drug site I: there the rotational correlation time was at least 72 ns. In the sites of the second type, this time was about 40 ns, and in the sites of the third type, about 10 ns. The faster was the rotation, the higher was the fluorescence quenching rate. Probably, it is this motion that is responsible for different fluorescence decay times in different HSA sites.     

Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperatures (?5 to 5°C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40° C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers.Whole erythrocytes were depleted of 30–50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3′-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot.The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.     

Kinetics of the rearrangement of the solvation shell of an excited fluorescent probe 4″-dimethylaminochalcone

A study was made of the processes associated with the quenching of 4″-dimethylaminochalcone (DMAC) fluorescence by proton-donor solvent (1-butanol). The kinetics of deactivation of the DMAC excited state was assessed by transient absorption spectra with a time resolution about 50 fs and by fluorescence decay with ～30-ps resolution. The following sequence of events could thus be envisaged: (i) the DMAC molecule in the ground state (prior to excitation) makes a hydrogen bond with an alcohol molecule; (ii) absorption of a light quantum causes a corresponding increase of the DMAC dipole moment; the H-bond is retained; (iii) the solvation shell formed by alcohol dipoles is reorganized in response to the raise of the DMAC dipole moment, with an energy expenditure about 24 kJ/mol and a time constant about 40 ps; the initial H-bond is still retained; (iv) processes leading to fluorescence quenching occur with an effective time constant of nearly 200 ps. Since quenching is far slower than solvate rearrangement, one can suppose that it is not a direct consequence of shell relaxation or prior H-bonding. Thus, DMAC fluorescence quenching may involve different processes observed with other aromatic molecules: H-bond rearrangement from a nonquenching to a more ‘efficient’ conformation, charge transfer between the excited molecule and alcohol, or solvent-induced out-of-plane twist of the DMAC amino group.     

Study of effects of cholesterol on the polar groups of the lipid bilayer using a fluorescent probe, 4-dimethylaminochalcone

Fluorescence parameters of a probe 4-dimethylaminochalcone (DMAC) in egg phosphatidylcholine bilayer lipid membranes are extremely sensitive to cholesterol, since the latter influences the solvation shells of DMAC formed by lipid molecules. The membrane population of DMAC is heterogeneous and is represented by two populations of molecules. The first of them contains about 60% of DMAC molecules, which fluoresce with a short (0.3 ns) decay time at 485 nm; their solvation shells are insensitive to 30 mol. % cholesterol. The second DMAC population is responsible for more than 50% of total fluorescence. Reorientation of the probe solvation shell dipoles is accompanied by a noticeable Stokes shift of the DMAC fluorescence spectrum; in the absence of cholesterol the Stokes shift amounts to 50 nm (i.e., from 485 to 535 nm). In the presence of 30 mol. % cholesterol, the effect of relaxation of the solvation shells on the DMAC fluorescence shift is reduced to 22 nm. At the same time, cholesterol reduces fluorescence quenching in the second population of DMAC molecules due to a twofold increase in their fluorescence quantum yield. Reorientation of solvation shell dipoles inducing the Stokes shift is complete within less than 0.1 ns, apparently, at the expense of fast-relaxing dipoles, viz., water molecules penetrating into the lipid bilayer. The data obtained suggest that cholesterol induces only partial modification of the lipid bilayer; some membrane regions are not exposed to its effect even when the cholesterol fraction constitutes up to 30% of the total lipid content.     

Fluorescent probe 4-dimethylaminochalcone: mechanism of fluorescence quenching in nonpolar media

The reasons for the high sensitivity of the fluorescent probe 4-dimethylaminochalcone (DMC) to nonpolar environment were explored. It was shown that, at room temperature, the fluorescence quantum yield in nonpolar media at 20 degrees C is lower than 0.01 (0.001 in methylcyclohexane). However, as temperature was lowered to -196 degrees C, the yield in methylcyclohexane increased more than 200 times. At the same time, the oscillator strength of absorption transition increased, and the absorption spectrum was shifted to red. These results, together with quantum chemistry calculations suggest that, for fluorescence quenching to occur, some barrier in the DMC molecule, probably the barrier of rotation about C-C bonds, should be overcome. In other words, the quenching is associated with the transition of DMC molecules from a flat conformation (energy minimum) to other, nonflat conformations through rotations about C-C bonds. The phosphorescence of DMC at low temperatures was detected. This suggests that fluorescence quenching is caused by radiationless transitions from the excited singlet level to the ground and triplet levels, and rotation about bonds facilitates these transitions.     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Rotational dynamics of immunoglobulins with fluorescent haptens on a membrane surface

The rotational dynamics of rabbit immunoglobulin G with fluorescent lipid haptens on a membrane surface has been studied by nanosecond fluorescence emission anisotropic spectroscopy. It has been found that the rotational angles of the antibody are very restricted on the membrane, but that the rotation rate itself is not appreciably lower than that in solution, and is independent of the membrane fluidity.     

Voltage sensitivity of the fluorescent probe RH421 in a model membrane system.

The voltage sensitivity of the fluorescent styrylpyridinium dye RH421 has been investigated in dimyristoylphosphatidylcholine vesicles by inducing an intramembrane electric field through the binding of the hydrophobic ion tetraphenylborate (TPB). To assess the probability of electrochromic and solvatochromic mechanisms for the dye response, the ground-state dipole moment of the dye in chloroform solution was determined from dielectric constant measurements to be 12 (+/- 1) Debye, and the change in dipole moment upon excitation was calculated from measurements of the Stokes shift in solvents of varying polarity to be 25 (+/- 11) Debye. As well as causing absorbance and fluorescence changes of membrane-bound dye, the TPB-induced electrical field was found to reduce significantly the pKa of the dye. The pH at which experiments are carried out is, thus, an important factor in determining the amplitude of the voltage-induced absorbance and fluorescence changes. The observed absorbance changes induced by the field are inconsistent with a pure electrochromic mechanism. A reorientation/solvatochromic mechanism, whereby the electrical field reorients the dye molecules so that they experience a change in polarity of their lipid environment is likely to make a significant contribution to both the spectral changes and to the field effect on the acid-base properties of the dye.     

Estimations of membrane potentials in Streptococcus faecalis by means of a fluorescent probe

Membrane potentials in $\text{Streptococcus faecalis (faecium)}$ were estimated by means of the fluorescent probe, 1,1′-dihexyl-2,2′-oxycarbocyanine. In the absence of D-glucose the potential was ?60 to ?70 mV for normal cells suspended in 0.09 M NaCl + 0.01 M Tris-HCl at pH 7.5. When metabolism was initiated by the addition of D-glucose the cells became hyperpolarized (internal becomes more negative). The new potential, ?130 to ?140 mV, was fully manifested 35 seconds after the glucose was added. N,N′-dicyclohexylcarbodiimide, a membrane ATPase inhibitor prevented the hyperpolarization seen upon the addition glucose. The results are consistent with the view that glycolyzing cells generate a considerasble electrical potential across the cell membrane.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.     

9-Aminoacridine as a fluorescent probe of the electrical diffuse layer associated with the membranes of plant mitochondria.

1. Mitochondria from Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices caused a quenching of the fluorescence of 9-aminoacridine when mixed in a low-cation medium (approximately 1 mM-K+) and addition of chelators further decreased the fluorescence. Salts released the quenching of the 9-aminoacridine fluorescence and the efficiency of the release appeared to be mainly dependent on the valency of the cation (C3+ greater than C2+ greater than C+). 2. The results are consistent with the theory of charge screening and demonstrate that 9-aminoacridine is a convenient probe of the behaviour of cations on the membranes of mitochondria and in the diffuse layer associated with these membranes. 3. The concentration of salt required to achieve half-maximal release of quenching of 9-aminoacridine fluorescence was proportional to the concentration of mitochondria in the solution and theoretical considerations show this effect to be inherent in the Gouy-Chapman theory. 4. 9-Aminoacridine was removed from the bulk of the solution by the mitochondria to a far greater extent than was Na+ or K+, which is suggested to be due to the formation of bi- and poly-valent cations by aggregation of 9-aminoacridine molecules in the diffuse layer. This would have implications for the use of 9-aminoacridine to determine delta pH across membranes. 5. Jerusalem-artichoke mitochondria removed from 9-aminoacridine and Ca2+ from the bulk of the solution and required more ions to screen the membranes than did an equal concentration (mg of protein/ml) of Arum mitochondria, indicating that Jerusalem-artichoke mitochondria contain more negative charges per mg of protein.     

Pyronin G as a fluorescent probe for quantitative determination of the membrane potential of mitochondria

Added to mitochondrial suspension, pyronin G changes the intensity of its fluorescence depending on the membrane potential (energy state) of the mitochondria. The mechanism of this effect is studied and a dependence is obtained between the membrane potential and the fluorescence intensity. This permits quantitative determination of the membrane potential by the changes in the fluorescence of the suspension. A method is proposed for measuring the membrane potential of vesicles in the -120 to -220 mV interval.