Decay-associated emission spectra and other spectral evidence for the physical intercalation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene into DNA

A benzo[a]pyrene derivative, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, forms physical complexes with DNA. The measured absorption spectrum of the hydrocarbon in the complex is shifted approximately 10 nm to the red and the fluorescence emission spectrum is red-shifted approximately 6 nm, characteristic of a physical intercalation complex. The decay-associated emission spectra of the hydrocarbon in the presence of DNA have been measured, thus providing a new technique to obtain information about the DNA binding sites. The decay-associated emission spectra of the free and bound hydrocarbons were obtained by deconvoluting the time-dependent emission at several wavelengths. Stern-Volmer plots with iodide and silver ions as quenchers suggest that at least one set of binding sites for the formation of a physical intercalation complex between the benzo[a]pyrene derivative and DNA is at guanine sites in the biopolymer.     

Salt-induced alterations of the fluorescence yield and of emission spectra in Chlorella pyrenoidosa

1. The addition of salts to the suspending medium induces a decreasein the yield of chlorophyll a fluorescence in normal and DCMU-poisonedintact algal cells of Chlorella pyrenoidosa. Potassium and sodiumacetate cause a pronounced lowering of the fluorescence at relativelylow concentrations (0.01–0.1 M). MgCl₂ and KCl cause asimilar lowering of fluorescence but at much higher concentrations(0.1–0.4 M). In contrast to sodium acetate, ammonium acetatedoes not cause any significant change in the fluorescence transient.
2. Unlike the case in isolated chloroplasts, MgCl₂ decreasestheratio of short wavelength (mainly system 2) to long wavelength(mainly system 1) emission bands in both DCMU poisoned and normalcells. Since these salt-induced changes do not appear to berelated to the redox reactions of photosynthesis, the saltsmight have caused a decrease in the mutual distance betweenthe two photosystems by changing the microstructure of the chloroplastsin vivo thereby facilitating the spillover of excitation energyfrom strongly fluorescent system 2 to weakly fluorescent system1.
3. The light induced turbidity changes in intact algal cells,asmeasured by the increase in optical density at 540 nm, isreducedin the presence of these salts. However, MgCl₂ producesthegreatest reduction while Na acetate the least, even thoughbothof these salts (at the concentrations used) cause largesuppressionof the fluorescence transient. Moreover, the lightinduced turbiditychanges were, essentially irreversible.
4. Ashigh concentrations of salts increase the osmotic potentialof the bathing medium, it seems that the osmotic changes aswell as the ionic changes in the intact algal cells are responsiblefor the fluorescence quenching and changes in the mode of excitationtransfer observed in this study. In the case of low concentrationsof salts (e.g., 0.1 M Na or K acetate) the effects are predominantlyionic, and in the case of very high concentrations of MgCl₂(0.4 M), the osmotic effects play a much larger role.

(Received July 30, 1973; )     

Fitting fluorescence emission spectra of probes bound to biological membranes

We show that fluorescence emission spectra for molecules containing the dansyl fluorophor can be accurately described as skewed Gaussians, and that spectra for dansyl probes bound to biological membranes can be resolved using least-squares techniques into two components, representing probe bound to the lipid and protein sites in the membrane.     

How aggregation and conformational scrambling of unfolded states govern fluorescence emission spectra

In a case study on five homologous alpha-amylases we analyzed the properties of unfolded states as obtained from treatments with GndHCl and with elevated temperatures. In particular the wavelength of the tryptophan fluorescence emission peak (lambda(max)) is a valuable parameter to characterize properties of the unfolded state. In all cases with a typical red shift of the emission spectrum occurring during structural unfolding we observed a larger magnitude of this shift for GndHCl-induced unfolding as compared to thermal unfolding. Although a quantitative relation between aggregation and reduction of the unfolding induced red shifts cannot be given, our data indicate that protein aggregation contributes significantly to smaller magnitudes of red shifts as observed during thermal unfolding. In addition, other properties of the unfolded states, most probable structural compactness or simply differences in the conformational scrambling, also affect the magnitude of red shifts. For the irreversible unfolding alpha-amylases studied here, transition temperatures and magnitudes of red shifts are strongly depending on heating rates. Lower protein concentrations and smaller heating rates lead to larger red shifts upon thermal unfolding, indicating that under these conditions the protein aggregation is less pronounced.     

Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8r0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550_FImag) and the red (F680_FImag) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550_FImage or F675/F525_380EX) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740_FImage) and the comparable 380EX ratio (F450/F730_380EX) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420_280EX) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.     

High resolution emission spectra of one second delayed fluorescence from chloroplasts

The first well resolved emission spectra of white light-illuminated spinach chloroplasts at room temperature show that one second delayed fluorescence occurs at 685 nm. We demonstrate that reabsorption of this delayed fluorescence induces the second (probably prompt) emission observed at 730 nm and which we identify with the photosystem I peripheral antenna system.     

The ultraviolet fluorescence spectra of DPN-linked isocitrate dehydrogenase from bovine heart.

The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.     

Corrected fluorescence excitation and emission spectra of phytoplankton: toward a more uniform approach to fluorescence measurements

Techniques for correction of fluorescence emission and excitationspectra of phytoplankton are described, which can be appliedin any commercially available spectrophotometer. The correctionof the emission spectrum is based on the measurement of a calibratedlight source. The excitation spectra are corrected by meansof a quantum counter solution that measures the spectral intensityof the excitation system and separate correction for wavelength-dependenteffects of the excitation optics. The correction proceduresgive technically corrected spectra, i.e. spectra that are freefrom wavelength dependent bias, but do not give absolute intensityvalues. Spectra that have been properly corrected for instrumentalwavelength dependencies are suitable for intercomparison, bothintra- and interlaboratory. Another application is the derivationof spectral data that will be obtained by other techniques thatmake use of fluorescence measurements, such as flow cytometry,remote sensing and in situ instruments. A necessary conditionis that the spectral response functions of these instrumentsmust be known. ¹Present address: AKZO, Arla-CRL, PO Box 9300, NL-6800 SB Arnhem,The Netherlands     

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Quenching of protein fluorescence by transient intermediates in the liver alcohol dehydrogenase reaction.

The addition of saturating concentrations of NAD-+ and alcohol to liver alcohol dehydrogenase in a stopped flow fluorimeter results in a triphasic quenching of enzyme fluorescence. A rapid quenching occurs with a rate constant of 300 to 500 s-minus 1, followed by a slower reaction at 50 to 100 s-minus 1, and ultimately followed by a very slow reaction. The addition of NAD-+ to enzyme in the absence of substrate causes a rapid quenching of enzyme fluorescence at 300 to 500 s-minus 1, with the same amplitude as the rapid phase in the presence of substrate. These studies demonstrate that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that proton release is probably coupled to the conformational change.     

A fluorescence and NMR relaxation study of thermally-induced conformational changes in liver alcohol dehydrogenase

Fluorescence and NMR relaxation studies have been performed on horse liver alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of a noncovalently bound apolar probe, 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS), indicate that a significant thermal transition occurs in the protein in the range of temperature 0-40 degrees C, and that there are different temperature-dependent forms of the enzyme. The transition between these forms is affected by the binding of specific ligands to the enzyme's active site. Time-resolved fluorescence studies of the two tryptophan residues in the enzyme suggest that this thermal transition occurs around tryptophan-314, which is buried near the intersubunit region. Binding of nucleotide to the enzyme causes a decrease in spin-lattice relaxation time, T1, which may result from a decrease in the number of water molecules bound to the protein. The observed results may be due to the interactions between the structural domains into which the monomer of the protein is folded.     

Quenching of the intrinsic fluorescence of liver alcohol dehydrogenase by the alkaline transition and by coenzyme binding

The fluorescence of alcohol dehydrogenase is quenched by the acid dissociation of some group on the protein having an apparent pKa of 9.6 at 25 degrees C. The pKa of this alkaline quenching transition is unchanged by the binding of trifluoroethanol or pyrazole to the enzyme or by the selective removal of the active site of Zn2+ ion. This indicates that the ionization of a zinc-bound water molecule is not responsible for the quenching. The binding of NAD+ to the enzyme causes a drop in protein fluorescence and an apparent shift in the alkaline quenching transition to lower pH. In the ternary complex formed with NAD+ and trifluoroethanol the alkaline transition is difficult to discern between pH 6 and pH 11. In the NAD+-pyrazole ternary complex, however, a small but noticeable fluorescence transition is observed with a pKa(app) approximately 9.5. We propose that the alkaline transition centered at pH 9.6 is not shifted to lower pH upon binding NAD+. Instead, the amplitude of the alkaline quenching effect is decreased to the point that it is difficult to detect when NAD+ is bound. We present a model that describes the dependence of the fluorescence of the protein on pH and NAD+ concentration in terms of two independently operating, dynamic quenching mechanisms. Our data and model cast serious doubt on the identification, made previously in the literature, between the alkaline quenching pKa and the pKa of the group whose ionization is coupled to NAD+ binding.(ABSTRACT TRUNCATED AT 250 WORDS)