Discrete bathochromic shifts exhibited by fluorescein ligand bound to rabbit polyclonal anti-fluorescein Fab fragments

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by 14 to 15 nm and/or energies of 1.5 kcal mol^–1 relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.     

Utraviolet adsorption and fluorescence of human thyroalbumin]

Some spectral properties of human thyroalbumin have been studied. Ultra-violet absorption of the aqueous solution of this protein has two maxima: at the wavelengths of 276 and 296--298 nm. Under the excitation by a monochromatic light with the wavelength of 280 nm the thyroalbumin has the fluorescence spectrum with the maximum at 430 nm and the quantum yeild of fluorescence about 5,4%. It has been established that thyroalbumin fluorescence consists of two components with the maxima at 385 and 450 nm. Moreover the "sortwave" component is principally attributed for by the presence do iodoamino acids.     

Combined blood cell counting and classification with fluorochrome stains and flow instrumentation.

A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.     

Characterization of indoles by thin-layer chromatography and in situ fluorometry

The characterization of microgram quantities of a number of naturally occurring and synthetic indoles through a combination of thin-layer chromatography and in situ fluorescence spectroscopy is reported. Instrumental detection limits of 0.03–0.05 μg of the indoles are possible using the native fluorescence of the indoles in the ultraviolet range, with excitation maxima in the range 285–310 nm and emission maxima in the range 345–360 nm. Spraying with a dilute acid solution (0.1 N H₂SO₄ in methanol) produces an additional pair of maxima, with excitation at about 350 nm and emission at about 450 nm. The presence of a polar compound such as sulfuric acid or dimethyl sulfoxide in the spray produces an enhancement of the indole fluorescence. The procedure should find application in the determination of indoles in biological samples.     

Increased levels of serum lipofuscin derived from 3-hydroxyanthranilic acid in patients with chronic renal failure

Normal Caucasian male sera incubated with 3-hydroxyanthranilic acid to generate soluble lipofuscin were studied together with unincubated serum samples from uremic Caucasian males, using the methods of Schwertner & Hawthorne in order to identify a fluorescent substance found by them to increase in uremic sera. Ethanol extracts of uremic sera, of normal sera containing this soluble lipofuscin and of same normal serum blanks were prepared. Reversed-phase thin-layer chromatograms of the extracts developed with methanol-water (40:60, v./v.), displayed one significant spot per sample, with RF values of 0.89 +/- 0.02. The spots showed blue fluorescence in 366 nm ultraviolet light. Aqueous solutions of the spots from uremic sera and from 3-hydroxyanthranilic acid-incubated normal sera produced closely similar fluorescence excitation shoulders and maxima at approximately 321 nm and emission maxima at 402 +/- 3 nm with significantly higher intensities than the normal. Thin-layer chromatograms of the ethanol extracts were also prepared on silica gel G developed with ethanol. The uremic, the 3-hydroxyanthranilic acid-incubated normal sera and the normal blank sera showed identical patterns in 366 nm light. The findings demonstrate that serum lipofuscin derived from 3-hydroxyanthranilic acid either in vivo or in vitro yields the fluorescent substance or component separated by ethanol extraction and reversed-phase thin-layer chromatography and that this serum lipofuscin present at low concentration in normal sera increases in uremic sera.     

Abiotic Photophosphorylation Model Based on Abiogenic Flavin and Pteridine Pigments

A model for abiotic photophosphorylation of adenosine diphosphate by orthophosphate with the formation of adenosine triphosphate was studied. The model was based on the photochemical activity of the abiogenic conjugates of pigments with the polymeric material formed after thermolysis of amino acid mixtures. The pigments formed showed different fluorescence parameters depending on the composition of the mixture of amino acid precursors. Thermolysis of the mixture of glutamic acid, glycine, and lysine (8:3:1) resulted in a predominant formation of a pigment fraction which had the fluorescence maximum at 525 nm and the excitation band maxima at 260, 375, and 450 nm and was identified as flavin. When glycine in the initial mixture was replaced with alanine, a product formed whose fluorescence parameters were typical to pteridines (excitation maximum at 350 nm, emission maximum at 440 nm). When irradiated with the quasi-monochromatic light (over the range 325–525 nm), microspheres in which flavin pigments were prevailing showed a maximum photophosphorylating activity at 375 and 450 nm, and pteridine-containing chromoproteinoid microspheres were most active at 350 nm. The positions and the relative height of maxima in the action spectra correlate with those in the excitation spectra of the pigments, which point to the involvement of abiogenic flavins and pteridines in photophosphorylation.     

光系统Ⅱ核心复合物的稳态荧光光谱

在83K和160K两个温度下,通过激发波长对荧光发射谱的影响研究了光系统Ⅱ中核心复合物的荧光光谱特性。用不同波长的光激发,核心复合物的发射谱的最大发射峰值不变,用480、489、495和507nm的光分别激发核心复合物,其光谱最大峰值处的荧光强度随不同激发波长下β-胡萝卜素分子的吸收强度的增大而降低,在长波长区域光谱的变化依赖于首先被激发的色素分子。所以,激发波长的不同影响着核心复合物中能量传递的途径。通过高斯解析,分析出核心复合物中至少存在有7组叶绿素a组分,它们是Ch1 a660,Ch1 a670,Ch1 a680,Ch1 a682,Ch1 a684,Ch1 a687和Ch1 a690。     

Purification and Characterization of a New Pigment from the Green Alga, Bryopsis maxima

Several, new, water-soluble pigments have been detected in thematured thalli of the green alga, Bryopsis maxima. Among thepigments, a major red one has been purified and characterized.The red pigment has absorption maxima at 237, 268, 331, 450,485 and 520 nm and a shoulder at 570 nm. Its fluorescence emissionspectrum has maxima at 659 and 730 nm. The pigment has minuscharge at the pH above 3.0 and is soluble in water and polarorganic solvents but not in nonpolar solvents. Its molecularweight was estimated to be 1,490. The infrared, N.m.r. and massspectra suggest that the pigment has an open tetra pyrrole structure. ⁵Present address: Department of Biochemistry, Nippon MedicalSchool, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan. (Received July 2, 1987; Accepted August 26, 1987)     

Fluorescent assay for riboflavin binding to cytochrome P450 2B4

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.8+/-1 microM. The fluorescence intensity of riboflavin was decreased with increasing the cytochrome P450 2B4 concentration, indicating the transfer of resonance excitation energy from riboflavin (energy donor) to the cytochrome P450 2B4 heme (energy acceptor). The data obtained are suggestive of the existence of riboflavin binding site(s) on the hemeprotein molecule.     

Trichromatic color vision in the salamander (Salamandra salamandra)

Spectral sensitivity functions were measured between 334 nm and 683 nm in Salamandra salamandra by utilizing two behavioral reactions: the negative phototactic response, and the prey catching behavior elicited by a moving worm dummy. The action spectrum of the negative phototactic response revealed 3 pronounced maxima: at 360–400 nm, at 520–540 nm, and at 600–640 nm. In the range around 450 nm, there was a reaction gap where sensitivity could not be measured. The action spectrum of the prey catching behavior was entirely different: maximal sensitivity was found at 500 nm and at 570 nm. Between 500 nm and 334 nm sensitivity decreased continuously for about 1 log unit (Fig. 6).Experiments under chromatic adaptation using the prey catching behavior indicate that the relatively high sensitivity in the ultraviolet range is not due to a separate ultraviolet photoreceptor, but is based on the responses of a photoreceptor maximally sensitive at about 500 nm.Color discrimination was tested by moving a colored worm dummy within a differently colored surround of equal subjective brightness. The salamanders were able to discriminate blue from green, and green from red (Fig. 10). The results can be explained by assuming a trichromatic color vision based on 3 photoreceptor types maximally sensitive around 450 nm, 500 nm and 570 nm (Fig. 12).     

Neutral fluorescence probe with strong ratiometric response to surface charge of phospholipid membranes.

We report on dramatic differences in fluorescence spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission.     

Characterization of the laser-induced blue, green and red fluorescence signatures of leaves of wheat and soybean grown under different irradiance

The blue, green and red fluorescence emission of green wheat ( Triticum aestivum L. var. Rector) and soybean leaves ( Glycine max L. var. Maple Arrow) as induced by UV light (nitrogen laser: 337 nm) was determined in a phytochamber and in plants grown in the field. The fluorescence emission spectra show a blue maximum near 450 nm, a green shoulder near 530 nm and the two red chlorophyll fluorescence maxima near 690 and 735 nm. The ratio of blue to red fluorescence, F450/F690, exhibited a clear correlation to the irradiance applied during the growth of the plants. In contrast, the chlorophyll fluorescence ratio, F690/F735, and the ratio of blue to green fluorescence, F450/F530, seem not to be or are only slightly influenced by the irradiance applied during plant growth. The blue fluorescence F450 only slightly decreased, whereas the red chlorophyll fluorescence decreased with increasing irradiance applied during growth of the plants. This, in turn, resulted in greatly increased values of the ratio, F450/F690, from 0.5 – 1.5 to 6.4 – 8.0. The decrease in the chlorophyll fluorescence with increasing irradiance seems to be caused by the accumulation of UV light absorbing substances in the epidermal layer which considerably reduces the UV laser light which passes through the epidermis and excites the chlorophyll fluorescence of the chloroplasts in the subepidermal mesophyll cells.     

Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems.

Two kinds of aequorin-type photoproteins, i.e., halistaurin and phialidin, and four kinds of modified forms of aequorin, i.e., products of acetylation, ethoxycarbonylation, fluorescamine-modification and fluorescein labelling, were prepared. The modified forms of aequorin were more sensitive to Ca2+ than was aequorin in their Ca2+-triggered luminescence reactions, whereas halistaurin and phialidin were less sensitive. The emission maxima of luminescence were all within a wavelength range 450-464 nm, except for fluorescein-labelled aequorin, which emitted yellowish light (lambda max. 520 nm). A new technique of measuring Ca2+ concentration is suggested.     

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence excitation spectra of drought resistant and sensitive genotypes of triticale and maize

An influence of soil drought (7 or 14 d) and 7 d recovery on changes of leaf fluorescence excitation spectra at wavelengths of 450, 520, 690, and 740 nm (F₄₅₀, F₅₂₀, F₆₉₀, F₇₄₀) for drought resistant and sensitive genotypes of triticale and maize was compared. In non-stressed plants the differences between maize and triticale were observed for F₄₅₀ and F₅₂₀, but not for F₆₉₀ and F₇₄₀. Drought caused the increase in F₄₅₀, F₅₂₀, and F₆₉₀ and this increase was more distinct for drought sensitive genotypes. After re-hydration, chlorophyll fluorescence mostly recovered to values of control plants. Drought caused significant increase in F₆₉₀/F₇₄₀ but not in F₄₅₀/F₆₉₀ and F₄₅₀/F₅₂₀. For triticale, highest increase in F₆₉₀/F₇₄₀ was observed in the 4^th and 7^th leaves of resistant genotype and contrarily in maize for the sensitive one. After recovery, the F₄₅₀/F₅₂₀, F₄₅₀/F₆₉₀, and F₆₉₀/F₇₄₀ ratios mostly returned to values of control plants.     

Distribution of UV-shielding of the epidermis of sun and shade leaves of the beech (Fagus sylvatica L.) as monitored by multi-colour fluorescence imaging

Plants can protect against damaging ultraviolet (UV) radiation by accumulating UV-absorbing substances in the epidermis of the leaves. Sun and shade leaves of a free standing beech tree (Fagus sylvatica L.) were studied for the differences in UV-shielding of the epidermis by means of multi-colour fluorescence images taken with UV and blue excitation. The distribution of the fluorescence intensity was detected over intact leaves in the emission maxima in the blue at 440 nm (F440), in the green at 520 nm (F520), in the red at 690 nm (F690) and in the far red at 740 nm (F740). Images of the logarithmic ratio between F690 excited in the blue and the UV (log (^BF690/^UVF690)) were calculated representing the relative absorption of UV in the epidermis and thus the degree of UV-shielding. It was found that UV-shielding is stronger for sun leaves than for shade leaves and better for the upper (adaxial) leaf side than for the lower (abaxial) leaf side of both leaf types. Within one leaf the highest value for the ratio log (^BF690/^UVF690) and thus the highest UV-shielding was found at the leaf rim which in broad leaves contains young tissue.     

Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

Study of interaction of cytochrome P450 2B4 with riboflavin by fluorescence spectrometry

Fluorescence quenching of riboflavin by cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent cytochrome P450 reductase, an essential electron carrier during cytochrome P450 catalysis. Fluorescence titration measurements revealed that cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of cytochrome P450 2B4.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.