Nanosecond time-resolved emission anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid

The fluorescence emission anisotropy of 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid has been studied using nanosecond time-resolved emission techniques. These studies demonstrate that the previously reported decrease in the steady state emission anisotropy, 〈r〉, with gestational age is due to a change in the rate of rotational motion of the probe. The emission anisotropy decays to a limiting value (r_∞) greater than zero, suggesting a hindered rotation of the probe, and this is independent of gestational age. The decay function for the emission anisotropy of amniotic fluids from 17, 29, 40 and 41 weeks in gestational age can be best expressed as a single exponential plus a constant term, with rotational correlation times varying from 17 ns to 2.2 ns, respectively. The zero time emission anisotropy remains approx. 0.30 for both early and late gestational times.     

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β-d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ_S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ_L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ_S and φ_L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.     

Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.     

Genetic characterization of a polymorphic murine cell-surface glycoprotein

As described in the preceding paper, monoclonal antibodies have been raised by immunization of rats with mouse hematopoietic cells which detect a major cell-surface glycoprotein (M_r=95 000) of mouse bone-marrow cells of the granulocytic series. While most of the monoclonal antibodies detect this molecule on bone-marrow and spleen cells of all mouse strains, two antibodies recognize alternative allelic forms of the molecule. One alloantigen is expressed in all the remaining inbred strains examined. The alloantigens are codominantly expressed on the cells of F₁ mice. Backcrosses of DBA/2 and C57BL/6 with F₁ mice (B6D2F₁) confirmed that a single genetic locus is involved in the expression of the two antigenic forms and demonstrated linkage to Ly-m11 which has previously been mapped to mouse chromosome 2. These genetic mapping experiments and the biochemical properties of the glycoprotein suggested that it might be identical to a glycoprotein first identified on murine fibroblasts by Hughes and August and designated Pgp-1. This has been firmly established by exchange of monoclonal antibody reagents and sequential immunoprecipitations.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

Nanosecond emission anisotropy of interacting enzymes aspartate aminotransferase glutamate dehydrogenase.

Nanosecond fluorescence studies were performed on mitochondrial aspartate aminotransferase from beef liver to determine whether the dimeric enzyme displays any modes of flexibility in the nanosecond range. The most informative quantities calculated from nanosecond fluorescence measurements S(t) and D(t) decay in a monoexponential manner with decay times τ_S = 13 and τ_D = 10 nanoseconds respectively. The observed rotational correlation time θ = 43 M-seconds yields a volume for the dimeric enzyme of 1.97 × 10⁵ A^o3. The rotational correlation time of aspartate aminotransferase is influenced by the presence of the enzyme glutamate dehydrogenase.     

Nanosecond spectroscopy of a dimeric enzyme: plasma amine oxidase.

The fluorescence dye 1-anilino-naphthalene-8-sulphonic acid (ANS) was used as a probe of non-polar binding sites in the enzyme plasma amine oxidase. Steady fluorescence measurements indicate that ANS binds to a single binding site of the dimeric enzyme with a dissociation constant of 5 microns. This binding site is different from the catalytic binding site. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 105 ns) corresponds to the rotation of a rigid dimeric macromolecule. A rotational correlation time of 120 ns was obtained with the enzyme labelled with pyrenebutyric acid. It is concluded that the dimeric enzyme does not exhibit any modes of flexibility due to independent rotation of the subunits in the nanosecond range.     

Nanosecond spectroscopy of retinol.

Nanosecond fluorescence spectroscopy was used to study the unique binding site of the retinol-binding protein (RBP) from human serum. At pH 7.4, the binding of retinol to RBP caused the following spectroscopic changes in the ligand: (a) an enhancement of the fluorescence decay time (gamma = 8 ns); and (b) an increase in the emission anisotropy (A = 0.29). Retinol in hexane has a fluorescent decay time of 4.2 ns and a low emission anisotropy (A = 0.02). The increase in the fluorescence decay time of bound retinol is not due to dielectric relaxation effects of polar groups, since nanosecond time-resolved emission spectra of either retinol in glycerol or retinol bound to RBP, failed to show any time-dependent shifts in emission maxima during the time period investigated 0 to 30 ns. The degree of rotational mobility of bound retinol was investigated by time emission anisotropy measurements. The observed rotational correlation time (theta = 7.2 ns) is consistent with a rigid compact macromolecule of 21,000 molecular weight.     

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay.

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes

Nanosecond decays of the fluorescence anisotropy, $\text{r}$, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.The relative contributions of the fast and the infinitely slow decaying component to the steady-state value, $\text{r}$, of the fluorescence anisotropy were very similar for artifical and biological membranes.Angles, θ, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with ‘microviscosities’, 〈η〉. An increase in 〈η〉 from 1.5 to 5.2 P in our systems was accompanied by a decrease in θ from 49° to 30° while the decrease in the mean motional relaxation times, $\text{φ}{}_{\mathrm{<mtext>f</mtext>}}$, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in ‘microviscosities’ of cholesterol-containing membranes ($\text{r}\text{> 0.15}$) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

Availability to monoclonal antibodies of antigenic sites of the α and β subunits in active,denatured or membrane-bound mitochondrial F1-ATPase

The binding of five monoclonal antibodies to mitochondrial F₁-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F₁ integrated in the mitochondrial membrane, for purified β-subunit and for purified F₁ maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-β monoclonal antibodies bound more easily to the β-subunit than to active F₁, and recognized equally active F₁ and F₁ integrated in membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F₁ and better exposed in the isolated β-subunit. In addition, unfolding F₁ by urea strongly increased the binding of one anti-β monoclonal antibody (14 D₅) indicating that this domain is at least partly shielded inside the β-subunit. One anti-α monoclonal antibody (20 D₆) bound poorly to F₁ integrated in the membrane, while the other (7 B₃) had a higher affinity for F₁ integrated in the membrane than for soluble F₁. Therefore, 20 D₆ recognizes an epitope of the α-subunit buried inside F₁ integrated in the membrane, while 7 B₃ binds to a domain of the α-subunit well exposed at the surface of the inner face of the mitochondrial membrane.     

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be D _r=(1.14±0.15)×10⁷ s^-1 at 22°C. The experiment is based on a cw argon ion laser, a microfluorimeter with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.     

Phase fluorimetric lifetime spectra. I. In algal cells at 77 K

A new method for decomposing fluorescence emission spectra into their elementary components, based on the simultaneous recording of fluorescence intensity and lifetime vs. the emission wavelength, has been applied to the spectra of algal cells at liquid nitrogen temperature. A model of Gaussian components fits both τ(λ) and F(λ) spectra with the same parameters. The fluorescence lifetimes have been measured by phase fluorimetry at two modulation frequencies: 29 and 139 MHz. The final Gaussian decomposition is able to describe both the 29 and 139 MHz spectra. The following conclusions concerning the fluorescence spectra of Chlorella cells at 77 K can be drawn. These conclusions are also valid with minor changes for the other examined species. (1) An overlapping of different emitting bands occurs in all the spectra; therefore, a direct lifetime reading from phase delay measurement necessitates measurements being made at several frequencies. (2) At the F_max fluorescence level, the lifetime values of the two emissions usually associated with variable fluorescence are 0.53 ns (for B′₁; λ peak 688 nm), and 1.46 ns (for B′₂; λ peak 698 nm); these lifetimes are shorter than those we have measured at room temperature (approx. 1.8 ns). (3) Superimposed on B′₁ and B′₂ and with approximatively the same peak location, two long-lifetime components (B″₁, 4.8 ns; B″₂, 5.6 ns) are present. Two hypotheses can be proposed to explain these emissions: (i) the long-lifetime components arise from subsets of chlorophyll a disconnected from the functional antenna by the cooling process; and (ii) charge recombination in reaction centers leads to delayed fluorescence. (4) In the λ > 710 nm region, two main bands are required to describe the so-called Photosystem I emission: B₃ (0.8 ns; λ peak 715 nm) and B₄ (3.3 ns; λ peak 724 nm). The former band, usually unresolved in the amplitude fluorescence spectra, is a specific finding from lifetime measurements and has been associated with the antenna core of Photosystem I. No additional information has been obtained for B₄. A supplementary small band (B₅, 0.40 ns; $\text{λ}\text{peak}\text{? 740}\text{nm}$) is necessary to take into account the frequency effect and the τ(λ) decrease in the λ > 740 nm spectral range.     

Identification and expression of kallikrein gene family in rat submandibular and prostate glands using monoclonal antibodies as specific probes

Panels of monoclonal antibodies to three vasoactive peptide-producing enzymes: tissue kallikrein, tonin and arginine esterase A were developed, characterized and used as probes for identification of tissue-specific expression. In addition, immunoblot analyses were performed, using monospecific monoclonal antibodies which did not show cross-reactivity to related-purified enzymes in enzyme-linked immunosorbant assay (ELISA), and radioimmunoassay. We obtained the following results. In rat submandibular gland extract, the expression of 38 kDa kallikrein, 32 kDa tonin, and 18 kDa heavy chain of esterase A was identified by monoclonal antibodies to kallikrein (V₄D₁₁), tonin (1F₁₁), and esterase A (5A₁₀, 6C₁₁, and 4B₁₂), respectively. In the prostate gland, a 32 kDa kallikrein-like protein was identified by monoclonal antibodies to esterase A (5A₁₀, 6C₁₁ and 4B₁₂) and by antibodies recognizing both tonin and esterase A (5A₅), but not by antibody to kallikrein (V₄D₁₁) or to tonin (1F₁₁, 1G₆) in Western blot analysis. The esterase A-like enzyme in the prostate gland was found within the cytoplasm of ductal epithelial cells by using monoclonal anti-esterase A antibody (5A₁₀) but not by employing anti-tonin antibody (1F₁₁). These results indicate that tissue kallikrein, tonin, and esterase A are all expressed in the submandibular gland, while only esterase A or an esterase A-like enzyme is expressed in the prostate gland. The specific monoclonal antibodies can be used as probes for the identification and expression of the kallikrein gene-family enzymes.     

Monoclonal Antibodies to and Immunoaffinity Purification of Choline Acetyltransferase from Bovine Brain

Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS-1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68-kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT.     

Low temperature spectral properties of subchloroplast fractions purified from spinach

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F_I, F_II, and F_III, corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at −196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F_II (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at −196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F_I, F_II, and F_III at −196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F_I showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F_II and F_III in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F_I and F_II caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at −196 C with an increase in the yield of fluorescence especially in the case of F_I.     

Novel ROSA26 Cre-reporter Knock-in C57BL/6N Mice Exhibiting Green Emission before and Red Emission after Cre-mediated Recombination

The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F₁ mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F₁ mice and R26GRR /Ins1-Cre F₁ mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).     

In vivo measurement of F420 fluorescence in cultures of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum could be followed by measuring the intensity of fluorescence directly in the culture vessel, avoiding conventional time-consuming extraction procedures of fluorescent coenzymes. The influence of light scattering by the bacteria was investigated. It could be shown, that light scattering had only little effect on the measurement of coenzyme F₄₂₀ fluorescence. However, culture fluorescence did not correlate to methanogenic activity, due to superposition of bacterial fluorescence by fluorescence from cell-free coenzyme which accumulates in the culture medium. By use of time-resolved laser spectroscopy, different fluorescence lifetimes were obtained for intracellular (1.0 ns) and extracellular (2.5 ns) components, respectively. A combination of this technique with photobleaching measurements for direct determination of F₄₂₀ content of bacteria in a culture is proposed.     

The static and dynamic behaviour of fluorescent probe molecules in lipid bilayers

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in multibilayers of dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) above their respective phase transitions. The finding that the order parameter 〈P₂〉 of the absorption moment is significantly higher than that for the emission moment for each probe is shown to arise from a tilt of the emission moment relative to the molecular symmetry axis. It is further shown that while the order parameter 〈P₂〉 is the same for both probes in DMPC bilayers, it is higher for TMA-DPH than for DPH molecules in POPC bilayers. Considerations of the order parameters 〈P₄〉, however, show that this difference can be ascribed solely to the higher fraction of DPH molecules lying with their axes parallel to the bilayer surface. Furthermore it is found that TMA-DPH molecules undergo slower reorientational motions than DPH molecules in the same bilayer system. Nevertheless the motion of both probe molecules is faster in DMPC than in POPC bilayers. The results indicate that TMA-DPH is a more useful probe than DPH in the systems investigated.     

Change in mobility of side chains due to neutralization of charged poly(L-lysine) with dansyl group

Poly(L -lysine) having dansyl (5-dimethylamino-1-naphthalene-sulfonyl) groups to its side chains was prepared. The fluorescence spectra and fluorescence anisotropy ratios of the dansyl (DNS) group were measured in various conditions. In aqueous solution the increase in emission intensity was observed reflecting the alkali-induced coil-to-helix transition. In aqueous-methanolic solutions with methanol content above 60 wt %, the poly(L -lysine) with DNS group (DNS-PLL) was probed to show α-helical conformation from CD spectra. With addition of alkali, the increase in fluorescence intensity of α-helical DNS-PLL and the drastic change in fluorescence anisotropy ratio were observed. In this case the rotational mobility of DNS probe decreases, gives a minimum at a certain concentration of added alkali, and then increases again up to approximately the initial level. At the concentration where the rotational mobility gives the minimum, intensity of scattered light gives a maximum. This shows that suppression of the mobility of DNS side chains is caused by the intermolecular aggregation of α-helical DNS-PLL. This concentration of added alkali corresponds to the midpoint of neutralization to charged side chains of the DNS-PLL. The interaction that causes aggregate of α-helical DNS-PLL is suggested to be the intermolecular hydrogen bonding between neutralized and unneutralized side chains. © 1994 John Wiley & Sons, Inc.