Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence

A novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a 'shuffling' procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One 'hybrid gene' was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red-shifted emission maximum at 616 nm.     

A terminal energy acceptor of the phycobilisome: the 75,000-dalton polypeptide of Synechococcus 6301 phycobilisomes--a new biliprotein

A rapid procedure is described for the isolation of "linker" polypeptides (Lundell, D. J., R. C. Williams, and A. N. Glazer. 1981. J. Biol. Chem. 256:3580-3592) of cyanobacterial phycobilisomes. The 75,000-dalton component of the core of Synechococcus 6301 phycobilisomes isolated by this procedure has been shown to carry a bilin similar in spectroscopic properties to phycocyanobilin. "Renatured" 75,000-dalton polypeptide has absorption maxima at 610 and 665 nm and a fluorescence emission maximum at 676 nm, similar to that of intact phycobilisomes. A complex of allophycocyanin and a 40,000- dalton bilin-carrying fragment of the 75,000-dalton polypeptide, obtained by limited tryptic digestion, is described. This complex, which lacks allophycocyanin B, shows a fluorescence emission maximum at 676 nm. The above data indicate that the 75,000-dalton polypeptide functions as a terminal energy acceptor in the phycobilisome.     

Fluorimetric microassay of DNA using a modified thiobarbituric acid assay

A method for the rapid, specific measurement of minute amounts of DNA is presented. The method is sensitive to about 20 ng of DNA, which is equivalent to approximately 3000 mammalian cells. The method employs enzymatic liberation of desoxyribose with DNase and phosphodiesterase. The liberated desoxyribose is reacted with thiobarbituric acid to yield a fluorescing compound with an excitation maximum at 532 nm and an emission maximum at 549 nm, measured with a fluorimeter. The entire procedure is accomplished within a few hours. The result is linear up to about 1 μg DNA or 3 × 10⁵ lymphocytes.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

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. Since 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.     

Colorimetric method for the assay of heparin content in immobilized heparin preparations

The properties of the metachromatic dye toluidine blue have been utilized to determine colorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations.     

Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase

We have measured the steady-state tryptophan fluorescence spectrum of cytochrome oxidase in its oxidized and fully reduced states. Reduction of the oxidized enzyme by sodium dithionite causes an apparent shift in the fluorescence emission maximum from 328 nm, in the oxidized enzyme, to 348 nm, in the reduced enzyme. This spectroscopic change has been observed previously and assigned to a redox-linked, conformational change in cytochrome oxidase [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. When dithionite-reduced enzyme sits in an open cuvette, the enzyme returns to the oxidized state, and the fluorescence maximum shifts back to 328 nm. However, the time course of the fluorescence change does not follow the redox state of the enzyme, monitored spectrophotometrically at 445,605, and 820 nm, but follows the disappearance of dithionite, which absorbs at 315 nm. Moreover, when the fluorescence emission spectrum of the dithionite-reduced enzyme is corrected for the absorbance due to dithionite, the fluorescence maximum is found 2 nm blue shifted, relative to that of the oxidized enzyme, at 326 nm. This dithionite-induced, red-shifted steady-state tryptophan fluorescence is also seen with the non-heme-containing enzyme carboxypeptidase A. The tryptophan emission spectrum of untreated carboxypeptidase A is at 332 nm, whereas in the presence of dithionite the emission spectrum of carboxypeptidase A is at 350 nm. When corrected for the absorbance of dithionite, the tryptophan emission maximum is at 332 nm. We have also used the photoreductant 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Tryptophan fluorescence studies of plasma fibronectin: effects of environmental factors

Steady state fluorescence measurements have been used to study tryptophan fluorescence of plasma fibronectin. The native protein has an emission maximum at 337 nm with a quantum yield of 0.03. A red shift of emission maximum was observed in 3–5M urea and a further red shift in 7–8M urea. The emission maximum shifted from 337 to 345 nm when the temperature was changed from 30 to 80°C, with a midpoint of thermal denaturation at 58°C. Similarly, the emission maximum shifted from 337 to 345 nm when the solution pH was increased from 9 to 12, with a midpoint of pH transition at 10.6. The results obtained from difference absorption spectroscopy studies suggest that the unfolding of fibronectin at alkaline pH is related at least in part to ionization of tyrosine residues. Since most of the tryptophan residues are in invariant positions in homology sequences, it is suggested here that tryptophan residues are useful intrinsic probes for elucidating fibronectin structure in solution.     

Selective,switchable fluorescent probe for heparin based on aggregation-induced emission

A positively charged tetraphenylethene (TPE) derivative, TPE-4MN, was synthesized as a probe for heparin based on aggregation induced emission. On the addition of 5.0 μg/mL of heparin, TPE-4MN showed an enhanced emission of about 10-fold. The change in fluorescence at 475 nm was linear over a range of heparin concentrations of 0–1.0 μg/mL with an R = 0.99988 and the limit of detection (LOD) was calculated to be 0.75 μg/mL. The mechanism of the detection was proven to be through an ion pairing interaction. TPE-4MN showed good selectivity for heparin over other types of polysaccharides and could easily distinguish heparin from heparan sulfate, a glycosaminoglycan having a similar structure to that of heparin.     

The fluorescent characterization of the polymerized microtubule-associated protein Tau

A new fluorescence formed while microtubule-associated protein tau was incubated at 25 and 37C for hours, with its maximum excitation at 230 and 280 nm, respectively. The fluorescence completely formed after tau was incubated in phosphate buffer and Tris-HCl buffer for approximately 20 h, with a relaxation phase about 2-4 h. The light scattering of the sample solution improved during formation of the fluorescence when tau was incubated. Both the fluorescence and tau oligomers did not form when tau was incubated in the buffers containing DTT. On the other hand, heparin improved both tau aggregation and the fluorescence formation. It suggests that the fluorescence comes from tau polymerization, which may follow the mechanism of tyrosine-tyrosinate emission for a protein not containing any tryptophan residues. This new fluorescence could be used as a probe to tau polymers.     

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ～650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.     

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.     

Detection of 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine) in the bovine brain by a fluorometric assay

A new sulfur imino acid, 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine), has been detected in the bovine brain by means of fluorometric and HPLC procedures. The fluorometric assay is based on the fluorescent property of the copper-ketimine interaction product at pH 11.5. Other ketimines do not fluoresce in these conditions. The fluorophore exhibits an excitation maximum at 353 nm and an emission at 462 nm and is stable for at least 24 h. In the test conditions the fluorescence is proportional to the ketimine concentration from 1 to 200 microM. Detection of endogenous lanthionine ketimine has been performed after a simple enrichment procedure (brain deproteinization and extraction with diethyl ether) which minimizes degradative by-reactions of the unstable ketimine. The concentration of this new sulfur imino acid in the brain ranges from 0.5 to 1 nmol/g in three different samples. Identification and quantitations were confirmed by an HPLC procedure which takes advantage of the selective absorption at 380 nm of the phenylisothiocyanate-ketimine adduct. The identification of lanthionine ketimine in nervous tissues may have important metabolic and physiological implications.     

Isolation and Function of Allophycocyanin B of Porphyridium cruentum

Allophycocyanin B was purified to homogeneity from the eukaryotic red alga Porphyridium cruentum. This biliprotein is distinct from the allophycocyanin of P. cruentum with respect to subunit molecular weights, and spectroscopic and immunological properties. The purified allophycocyanin B has a long wavelength absorption maximum at 669 nm at room temperature and at 675 nm at −196 C while the fluorescence emission maximum is at 673 nm at room temperature and 679 nm at −196 C. The emission spectrum of allophycocyanin shifted only 1 nm, from 659 to 660 nm, on cooling to −196 C, and was the same with allophycocyanin crystals as it was with pure solutions of the pigment. Phycobilisomes from P. cruentum have a major fluorescence emission band at 680 nm at −196 C which emanates from the small amount of allophycocyanin B present in the phycobilisomes. Light energy absorbed by the bulk of the biliprotein pigments is transferred to allophycocyanin B with high efficiency.     

Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ)₆ hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.     

Excitation spectra for Photosystem I and Photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis.

The fluorescence of variable yield at 750 nm at −196 °C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at −196 °C at the minimum, F_O, level and the maximum, F_M, level of the emission at 750 nm. The difference spectrum, F_M–F_O, which represents the excitation spectrum for F_V is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex.

Fluorescence at the F_O level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, , which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of F_O/F_V measured at 692 nm and the extent of F_V at 750 nm. According to this procedure the excitation spectrum of Photosystem I at −196 °C was determined by subtracting 1/3 of the excitation spectrum of F_V at 750 nm from the excitation spectrum of F_O at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex.

The wavelength dependence of was determined from fluorescence measurements at 692 and 750 nm at −196 °C. is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Investigations of the Blue-green Fluorescence Emission of Plant Leaves

The UV light (337 nm) induced blue-green fluorescence emission of green leaves is characterized at room temperature (298 K) by a maximum near 450 nm (blue region) and a shoulder near 525 nm (green region) and was here also studied at 77 K. At liquid nitrogen temperature (77 K) the blue (F450) and green fluorescence (F525) are much enhanced as is the red chlorophyll fluorescence near 735 nm. During development of green tobacco leaves the blue fluorescence F450 (77 K) is shifted towards longer wavelengths from about 410 nm to 450 nm. The isolated leaf epidermis of tobacco showed only slight fluorescence emission with a maximum near 410 nm. The green fluorescence F525 was found to mainly originate from the mesophyll of the leaf, its intensity increased when the epidermis was removed. The red chlorophyll fluorescence emission was also enhanced when the epidermis was stripped off; this considerably changed the blue/red fluorescence ratios F450/F690 and F450/F735. The epidermis, with its cell wall and UV-light-absorbing substances in its vacuole, plays the role of a barrier for the exciting UV-light. In contrast to intact and homogenized leaves, isolated intact chloroplasts and thylakoid membranes did not exhibit a blue-green fluorescence emission.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.