Steady-state Fluorescence Emission Research Articles

Hydration and protein substates: Fluorescence of proteins in reverse micelles

Abstract The fluorescence properties of indole derivatives, lysozyme and azurin were investigated in reverse micelles of detergent sodium bis[2-ethylhexl]sulfosuccinate (Aerosol OT)* in n-hexane. L-tryptophan, l-methyl-tryptophan and n-acetyl-l-tryptophanamide exhibited complex fluorescence decays in reverse micelles. Fluorescence decays were best described using Gaussian bimodal distributions of lifetimes. Increasing hydration levels in the micelles resulted in a decrease in decay heterogeneity, as indicated by a large decrease in lifetime distribution widths. Steady-state polarization and fluorescence emission measurements indicated both an increase in average polarity of the environment around the indole derivatives and an increase in the mobility of the probes with increasing hydration levels. The fluorescence decays of lysozyme and azurin in reverse micelles were also found to be very complex and were described with Gaussian lifetime distributions. Increasing water content in the micelles caused marked decreases in both center and width of the lifetime distributions for these two proteins. Steady-state polarization measurements as a function of the extent of hydration revealed an increase in the average rotational rates of the tryptophan residues in lysozyme upon increasing water content. Thus, static polarization and lifetime measurements indicate that the amount of water present in the micelle may influence the amount of structural flexibility of the polypeptide chains and the rates of interconversion between conformational substates.

The purification and characterization of arginase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.

Spectroscopic studies of cyanobacterial phycobilisomes lacking core polypeptides

Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR6) genes encoding two highly conserved phycobilisome core polypeptides, a small linker polypeptide (LC8, apcC) and the allophycocyanin-B alpha-subunit (alpha APB, apcD), respectively, were interrupted by insertion of restriction fragments carrying the neomycin phosphotransferase gene of Tn5. The interrupted genes were used to transform Synechococcus sp. PCC 7002 to kanamycin resistance. The apcC- mutant assembled phycobilisomes lacking the LC8 polypeptide and the apcD- mutant assembled phycobilisomes lacking alpha APB. No other differences between the compositions of the mutant and wild-type phycobilisomes were detected. The apcC- strain grew about 25% more slowly than the wild-type, and its phycobilisomes dissociated more rapidly in 0.33 M Na/K-PO4 (pH 8.0) or in 0.75 M Na/K-PO4 at pH 8.0, at 40 degrees C, than did those of the wild-type. The phycobilisomes of this mutant were indistinguishable from those of the wild-type with respect to absorption and circular dichroism spectra, as well as time-resolved fluorescence emission. Steady-state emission spectra indicate a small decrease in long wavelength (680 nm) emission from the apcC- phycobilisomes and a complementary increase in shorter wavelength (665 nm) emission, relative to wild-type phycobilisomes. Strain apcD- phycobilisomes appear to be functionally indistinguishable from those of the wild-type, in spite of the absence of the two alpha APB subunits which bear terminal acceptor bilins. The only spectroscopic difference was seen in the steady-state fluorescence emission, for which the emission of the mutant was about 15% higher than that of the wild-type and was slightly blue-shifted. A phenotype has yet to be found for the apcD- mutation.

Collisional dynamics of the IF B 3Π(0+) state. III. Vibrational and rotational energy transfer

Rate constants at T=300 K for collision induced vibrational and rotational energy transfer within the IF B 3Π(0+) state have been determined using both cw and pulsed laser induced fluorescence. State-to-state vibrational energy transfer rate coefficients have been measured for IF(B; v′) collisions with He, Ne, Ar, Kr, Xe, N2, O2, and F2. Vibrational energy transfer has also been studied as a function of the initially excited vibrational level. Steady-state fluorescence emission from v′=3 has been analyzed to yield total rotational removal rate coefficients with the noble gases and N2 as collision partners. Vibrational energy transfer is generally inefficient; the thermally averaged cross sections for single quantum transfer (Δv=−1) from v′=3, σ(3,2), decrease from 1.2% to 0.3% of the gas kinetic cross sections, σg, for the periodic series He to Xe. For diatomic collision partners, the respective values of σ(3,2)/σg for N2, O2, and F2 are 0.014, 0.033, and 0.020. The Δv=−1 cross sections for the noble gases monotonically decrease with μ1/3 while no clear dependence on the collision reduced mass is observed with N2, F2, and O2. The vibrational transfer cross sections for the noble gases, N2, and O2 scale linearly with vibrational quantum number. The results also reveal that ‖Δv‖=1 transfer is at least an order of magnitude more probable than that for ‖Δv‖=2. Rotational energy transfer is the most efficient kinetic process in IF(B). The estimated efficiencies for total rotational energy transfer from J′=22 in v′=3 with the rare gases and N2 are typically 20 to 200 times greater than those for vibrational transfer. The rate coefficients range from (9.7±1.8)×10−11 cm3 molecule−1 s−1 for He to (1.1±0.1)×10−10 cm3 molecule−1 s−1 for Xe. The rotational transfer rate coefficients show a smooth dependence on both the collision reduced mass and the polarizability of the collision partner. The qualitative results of these experiments are discussed in relation to traditional energy transfer models.

APUD cell development in grafts from chick or quail presumptive enteric endoderm combined with a fragment of notochord : E.L.M.J. Wiertz-Hoessels and J. Drukker. Department of Anatomy/Embryology, Medical Faculty, University of Limburg, Maastricht, the Netherlands

Characterization of the internal environment of a sol–gel matrix is an important area of investigation in optical biosensors. In the present study, different sol–gel compositions were prepared by varying the water (H2O) to tetraethyl-orthosilicate (TEOS) ratio (R) from 1 to 16 and the changes in the internal environment of the sol–gel both in bulk and thin films as a function of aging (storage) were investigated using fluorescence spectroscopy. We focussed on the fluorescence characteristics , viz. emission and excited state lifetime of Hoechst 33258 (H258), a bisbenzimidazole derivative, which was used as fluorescence probe entrapped in the TEOS derived sol–gel bulk and thin films. These sols were prepared at a low pH (∼2.0) and the thin films were coated by dip coating technique at withdrawal speeds of 1 cm/min and 0.1 cm/min. Usually, uniform thin films were obtained at a high speed (1 cm/min) and partially cracked film at a low speed (0.1 cm/min) as observed by fluorescence microscope. These observations did not change during aging. On the contrary, three months long observations on steady-state fluorescence emission measurements on H258 depicted a blue shift from 535 nm to 508 nm at R = 1 in the sol–gel bulk, whereas at higher ratios this was not prominent. At all ratios, dual emission bands were observed in thin films. This may be due to faster sol–gel to xerogel transition during aging depending on the ratio (R). Analysis of the excited state decay profiles of H258 revealed a double exponential fitting having a short (τ1) and a long (τ2) component in both fresh and during aging, in the sol–gel bulk and thin films, indicating heterogeneity in the internal environment. The value of τ1 increased from 0.4 ns to 1.2 ns whereas τ2 attained a value from 3.0 ns to 3.6 ns at R = 1 upon aging in the sol–gel bulk. The corresponding values of τ1 and τ2 in thin films were 0.3 ns and 3.5 ns, respectively. The values of these decay components in thin films did not alter much due to storage, but their relative contributions showed more systematic changes in the thin films. The observed changes could be correlated to rigidification in the bulk depending on the ratio (R). This process was very slow at R ≥ 4. The heterogeneity in the internal environment of bulk and thin films upon aging appeared to be different as revealed from analysis of excited-state lifetime. Thus, the bisbenzimidazole derivative H258 appears to be very useful probe for characterizing the internal environment of both the sol–gel bulk and thin films.

The use of site-directed mutagenesis and time-resolved fluorescence spectroscopy to assign the fluorescence contributions of individual tryptophan residues in Bacillus stearothermophilus lactate dehydrogenase

Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (< 0.3 ns). Trp-203 is responsible for 75–80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.

Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer.

Data on the wavelength and temperature dependence of both time-resolved and steady state fluorescence emission are presented for allophycocyanin (AP) and for a crosslinked allophycocyanin trimer (XL-AP) (Ong LJ and Glazer AN: Physiol Veg 23:777-787, 1985). AP dissociates at high dilution and is not stable above 40 degrees C even at moderate protein concentration. In contrast, XL-AP does not dissociate even at very low protein concentrations and is completely stable up to 60 degrees C in the presence of 0.75 M NaK-phosphate, pH 7.0. The results show that XL-AP is superior to AP for use in conjugates that absorb and emit in the red region of the spectrum. The high stability of XL-AP at elevated temperatures at high phosphate concentrations suggests that this derivative may be useful in conjunction with nucleic acid probes.

Effect of trivalent lanthanide cations on chlorophyll fluorescence and thylakoid membrane stacking

We have used trivalent lanthanide metal cations in the buffering media of pea chloroplasts to probe the stacking arrangement of thylakoid membranes and the spatial distribution of chlorophyll-protein complexes of Photosystems I and II. Measurements of steady-state chlorophyll fluorescence emission spectra of pea chloroplasts at room temperature demonstrate that, within this tripositive valency group, the extent of membrane appression is a function of hydrated metal ionic radius. These results are in agreement with a recent investigation using monovalent and divalent metal cations (Karukstis, K.K. and Sauer, K. (1985) Biochim. Biophys. Acta 806, 374–389). In addition, the lanthanide cation concentration effective in producing the maximum chlorophyll fluorescence intensity upon grana formation is dependent on hydrated ionic size. The current investigation supports the proposed hypothesis that cation screening ability defines the extent of intermembrane separation as well as the extent of lateral distribution of chlorophyll-protein complexes.

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the flurophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at β93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, −146) emission maximum 333 nm; sperm whale (Tyr-103, −146, −151) emission maximum 331 nm. Science 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.

Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.

In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3‐(3,4‐DICHLOROPHENYL)‐1,1‐DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q‐ with stored oxidizing charges on the donor side of system II.At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants kα and kβ. The biphasic nature suggested a sequential two‐electron reduction of the primary acceptor Q, or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH2OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH2OH, and the flash induced state Sn of the water splitting enzyme, control the values of kα and kβ by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.

Fatty acids as model systems for the action of Ricinus leaf extract on higher plant chloroplasts and algae

The ability of long-chain unsaturated fatty acids to serve as models for the action of the protein fraction of Ricinus leaf extract on chloroplasts (and algae) has been determined experimentally for several parameters. These include steady-state fluorescence emission and excitation (measured at −196 °), fluorescence induction, light-induced absorption changes of chlorophylls a II and a I, Hill activity, and chloroplast ultrastructure. In addition, the sequential inhibition of system II- and system I-associated electron flow has been demonstrated as a function of increasing concentration of exogenous fatty acid.