Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase

Time-resolved fluorescence studies on the emission of NADH bound to porcine heart mitochondrial malate dehydrogenase [S)-malate:NAD+ oxidoreductase, EC 1.1.1.37), in the presence and absence of saturating levels of hydroxymalonate, were carried out. The lifetime of NADH bound in the ternary complex was determined to be 9.5 ns compared to 1.74 ns as reported in the literature. Steady-state and dynamic polarization data indicated a Debye rotational relaxation time in the range of 106-109 ns for the dimeric enzyme. This value is significantly larger than that calculated for a spherical protein and is consistent with the asymmetric dimer found by crystallographic studies.     

The structure of DAPI bound to DNA

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.     

Time-resolved fluorescence of conifers exposed to environmental pollutants

Summary With regard to an early diagnosis of defects within the photosynthetic system of conifers by air pollutants, we measured the chlorophyll fluorescence from microscopic parts of individual pine and spruce needles. In particular, different spruces had been exposed before to well-defined doses and concentrations of ozone and SO₂. In addition to spectral distributions and time courses of fluorescence intensity, fluorescence lifetimes were detected after picosecond laser pulse excitation. Time constants of ₁ = (0.10 ± 0.02) ns and ₂ = (0.50 ± 0.10) ns were measured for intact photosynthesis; an increase of ₂ and a further time constant ₃ = (2.5 ± 0.5) ns were found, if defects within the photosynthetic system occurred. Most significant defects were so far measured after exposition to high ozone doses during longer time periods.Paper presented at the Conference on Chlorophyll Fluorescence in Ecology, February 20–21, 1986, Hannover, FRG     

DAPI fluorescence in nuclei isolated from tumors.

In DNA histograms of some human solid tumors stained with nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.     

Time-resolved fluorescence of apoferritin and its subunits

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.     

Time-resolved extrinsic fluorescence of aromatic-L-amino-acid decarboxylase

The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.     

Time-resolved fluorescence spectroscopy of spinach chloroplast

Picosecond fluorescent kinetics and time-resolved spectra of spinach chloroplast were measured at room temperature and low temperatures. The measurement is conducted with 530 nm excitation at an average intensity of 2 · 10¹⁴ photons/cm², pulse and at a pulse separation of 6 ns for the 100 pulses used. The 685 nm fluorescent kinetics was found to decay with two components, a fast component with a 56 ps lifetime, and a slow component with a 220 ps lifetime. The 730 nm fluorescent kinetics at room temperature is a single exponential decay with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K, while the 685 and 695 nm fluorescent kinetics were unchanged. The time-resolved spectra data obtained within 10 ps after excitation is consistent with the kinetic data reported here. A two-level fluorescence scheme is proposed to explain the kinetics. The effect of excitation with high light intensity and multiple pulses is discussed.     

DAPI fluorescence of plant chromosomes prestained with actinomycin D

Two plants, Ornithogalum caudatum and Scilla siberica, with contrasting types of heterochromatin, were employed to investigate the effect of prestaining with actinomycin D on the differential DAPI fluorescence in nuclei and chromosomes. Actinomycin D-pretreated preparations exhibited in both plants a significantly lower overall fluorescence intensity than DAPI controls. In O. caudatum the fluorescence properties of DAPI-bright heterochromatin remained qualitatively unchanged, but in S. siberica chromosomes the differential fluorescence of DAPI-negative heterochromatin disappeared or occasionally was reversed. It is shown that the observations cannot be interpreted simply as a result of base specific drug-DNA interaction.     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.     

Time-resolved fluorescence spectroscopy of hematoporphyrin-derivative in human lymphocytes

This paper reports on time-resolved microfluorimetric measurements on hematoporphyrin-derivative (HpD)-treated lymphocytes. HpD is at present widely used as a tumor-locating and photosensitizing drug. It is therefore of great importance to study the extent to which the HpD uptake process depends on cell functional and structural properties. Time-resolved fluorescence measurements in single cells are very useful in this respect, since they give information on the content of fluorescent molecules through fluorescence peak-intensity, and, indirectly, on the binding properties through the fluorescence decay times. In particular, we studied the dependence of HpD fluorescence on the cellular functional state. To this end, we performed in-cell fluorescence measurements on human lymphocytes, both in quiescent conditions and in the pre-replicative phase, after stimulation with phytohemagglutinin (PHA). We found a higher HpD content in stimulated lymphocytes. Moreover, we found a spectral band around 575 nm, corresponding to a particular porphyrin species, in which the differences between normal and stimulated lymphocytes are more striking. The porphyrin species emitting in this band seems to play a role in the specific interaction of HpD with tumors, since a similar emission band has also been found in tumor cells containing HpD.     

Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy

Proton 2D-NOE spectroscopy has been used to investigate the three-dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA-dT) · poly(dA-dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG-m⁵dC) · poly(dG-m⁵dC) in low salt (0.1M NaCl) are righthanded B-structures. Poly(dG-m⁵dC) · poly(dG-m⁵dC) in Mg²⁺ (3 mM) solution exhibits a pattern characteristic of the left-handed Z-form. These results for poly(dA-dT) · poly(dA-dT) are in contrast to suggestions that this copolymer exists as a left-handed form, either in low or high salt. We present pure absorption-mode 2D-NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.     

Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was investigated as a cholesterol analogue to examine sterol domains in and spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Fluorescence lifetime, acrylamide quenching analyses, and intermembrane exchange kinetics were consistent with the presence of at least two sterol domains in POPC. Fluorescence lifetime was determined by phase and modulation fluorescence spectroscopy and analyzed by nonlinear least-squares as well as continuous distributional analyses. Both methods demonstrated that pure dehydroergosterol in POPC SUV had two lifetime components (C) and fractional intensities (F) near C1 = 0.851 ns (F1 0.96) and C2 = 2.668 ns (F2 0.004). In contrast to component C1, the center of lifetime distribution, fractional intensity, and peak width of dehydroergosterol lifetime component C2 was dependent on the polarity of the medium and vesicle curvature. The sterol domain corresponding to dehydroergosterol component C2 was preferentially quenched by acrylamide. Acrylamide quenching of dehydroergosterol fluorescence demonstrated that the two lifetime components of dehydroergosterol were not due to transbilayer sterol domains with different lifetimes. In a spontaneous exchange assay not requiring separation of donor and acceptor SUV, the lifetime component C2, but not C1, shifted to a shorter lifetime with altered distributional width. The kinetics of these lifetime and distributional width changes best fitted a two-exponential function, with a fast exchange rate constant K1 = 0.0325 min-1, t1/2 = 21.3 min, and a slow rate constant k2 = 0.00275 min-1, t1/2 = 261 min. The fast exchanging pool correlates with the longer lifetime component C2. These kinetics were confirmed both by dehydroergosterol exchange measured with fluorescence intensity and by [3H]cholesterol exchange. In summary, lifetime, distributional width, acrylamide quenching, and classical exchange assay data are consistent with the presence of at least two pools of sterol in POPC SUV.     

Selective excitation of mithramycin or DAPI fluorescence on double-stained cell nuclei and chromosomes

Summary Fluorescence spectra of leukocytes stained by both mithramycin and DAPI showed that the fluorescence of the two dyes can be separated efficiently by using different excitation wavelengths, for instance the 435 nm and the 365 nm mercury lines. In human chromosomes the complementary (reverse) banding pattern produced by these dyes may thus be observed on double stained chromosome spreads. In plants, for instance in Anemone blanda, the two dyes may reveal two different banding patterns. The results of absorption and fluorescence measurements suggest the existence of at least two binding sites, or types, for each dye, with different fluorescent yields and binding strengths.     

Time-resolved fluorescence study during denaturation and renaturation of curcumin-myoglobin complex

Curcumin influences the transition point, the concentration of denaturant required to effect 50% of the total change, of myoglobin denaturation. Curcumin enhances absorbance of myoglobin at 280 nm with a binding constant K=3.0×10(4) M(-1) whereas fluorescence of curcumin is quenched by myoglobin with a Stern-Volmer association constant of 2.5×10(5) M(-1). Unfolding process of myoglobin-curcumin induces a recovery in fluorescence lifetime loss. The gain in time-resolved fluorescence lifetime during unfolding has been again lost during refolding of curcumin-myoglobin complex by dilution process suggesting partial reversibility of unfolding process for both myoglobin and curcumin-myoglobin complex.     

Time-resolved fluorescence anisotropy of fluorescent-labeled lysophospholipid and taurodeoxycholate aggregates.

Previous work from this laboratory demonstrated that the environment-sensitive lysolipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- monomyristoylphosphatidylethanolamine (N-NBD-MPE), at concentrations below its critical micelle concentration (CMCN-NBD-MPE = 4 microM), reached maximum fluorescence yield upon the addition of taurodeoxycholate (TDC) at concentrations well below its CMC (CMCTDC = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concentrations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to determine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) were measured for N-NBD-MPE at the submicellar concentration of 2 microM, indicating a lack of interaction between the two molecules at this concentration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Based on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi(0.19-0.73 ns) was ascribed to local probe motions, whereas the longer phi was in a time range expected for global rotation of aggregates the size of simple bile salt micelles (3-6.5 ns). From the longer phi, molecular volume and hydrodynamic radii were calculated, ranging from approximately 15 A at 1 mM to approximately 18 A at 5 mM TDC. These data support the conclusion that monomeric lysolipids in solution seed the aggregation of numerous TDC molecules (aggregation number = 16 at 1 mM TDC) to form a TDC micelle with a lysolipid core at concentrations below which they both self-aggregate.     

Time-resolved spectroscopy of the blue fluorescence of spinach leaves

When excited by ultraviolet radiation, leaves of a great number of species of higher plants exhibit emission of blue fluorescence, comparable in intensity to the red emission of chlorophyll. The fluorescence decay of the blue emission of spinach leaves recorded by single photon counting techniques is decomposed into exponential components and it is shown that at least three different components are present. The lifetime of the three components does not show significant variations with the excitation or emission wavelengths. The excitation and emission spectra of each component were determined. The nature of the chemical compounds which cause this emission is discussed in relation to these spectra.