Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.     

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.     

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.     

Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties

Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.     

B-Phycoerythrin from Rhodella violacea: characterization of two isoproteins.

Two isoproteins of the "native" B-phycoerythrin of the red alga, Rhodella violacea, were purified from crude extracts by preparative polyacrylamide gel electrophoresis and subsequently characterized. The slower moving pigment in gel electrophoresis was designated B-PE I, the faster as B-PE II. Both were found to occur in about equal amounts. B-PE I has a molecular weight of about 280000 and an IEP at 4.39, B-PE II a molecular weight of nearly 265000 and an IEP at 4.23. B-PE I and II are characterized by absorption maxima at 568 and 542 nm and a shoulder at 500 nm in the visible part of the absorption spectra. Their absorption coefficients at 542 nm differ with values of 5.54 and 5.63, respectively. The fluorescence emission spectra show a single maximum at 575 for B-PE I and at 578 nm for B-PE II. Both spectra have a shoulder at 630 nm. The fluorescence yield of B-PE II is lower by 25%. In calibrated SDS gel electrophoresis of the purified pigments B-PE I and II show two subunits of molecular weights of 18900 and 29200 and 18500 and 29900, respectively. Quantitative amino acid analyses indicated, that the isoproteins are very similar. B-PE II, however, has a significantly higher content of acidic amino acids and a lower percentage of basic residues, which is in keeping with its lower isoelectric point. Functional aspects of the occurrence of two isoproteins of B-phycoerythrin are discussed.     

A high molecular weight terminal pigment (“anchor polypeptide”) and a minor blue polypeptide from phycobilisomes of the cyanobacterium Nostoc sp. (MAC): Isolation and characterization

A 94 kD pigment-polypeptide, which is presumed to be involved in anchoring the phycobilisomes to the thylakoids, was isolated from Nostoc phycobilisomes by gel filtration in 63 mM formic acid. The isolation condition did not require detergents or denaturating reagents, as in previous procedures, and enzymatic degradation was not observed at the low pH of 2.5. The anchor polypeptide thus obtained had absorption (Abs) and fluorescence maxima (Em) at 658 and 673 nm, respectively, in 63 mM formic acid at room temperature. The maxima shifted to longer wavelengths in 100 mM potassium phosphate (pH 6.8), Abs 665 and Em 683 nm at room temperature, and Abs 665 and Em 684 nm at liquid nitrogen temperature. The fluorescence maxima at both temperatures correspond to the longest wavelength component resolved in phycobilisomes from second derivative spectra. A minor blue polypeptide was also found by this isolation method. The molecular weight of this polypeptide was ca. 18,000 and is probably similar to a polypeptide which has been found in the phycobilisome core of other cyanobacteria.Abbreviations used -APB -subunit of allophycocyanin B - APC allophycocyanin - kD kilodalton - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PBS phycobilisome - PE phycoerythrin - PC phycocyanin - PS pigment system     

Characterization of the fluorescence of the antitumor agent, mitoxantrone

Studies have been conducted on the absorption, fluorescence excitation and fluorescence emission of mitoxantrone, an important antineoplastic agent. Mitoxantrone has been found to fluoresce with excitation maxima at 610 and 660 nm and emission maximum at 685 nm. Further characterizations of the fluorescence were undertaken to study its usefulness in biological studies. Mitoxantrone fluorescence intensity is altered by pH and the emission spectrum is red-shifted by DNA. Furthermore, the fluorescence polarization is enhanced by DNA, confirming the binding of the antitumor agent to DNA. The fluorescence spectra are slightly modified by changes in ionic strength and the addition of albumin. Data establishing the usefulness of fluorescence to measure serum concentrations in the range of 0.0 to 100 nM are presented. Such determinations can distinguish serum mitoxantrone from its non-fluorescent primary metabolite.     

Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde

Summary The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.     

Absorption and Fluorescence Spectra of Euchrysine Ggnx and Acridine Orange; Effects of Heparin, NaCl and a Cation Exchange Resin

The absorption and fluorescence spectra of two samples of dye labeled euchrysine were found to differ. One sample, labeled GGNX, had absorption and fluorescence maxima of 435 and 515 nanometers (nm) respectively. The other sample was not further labeled, but had absorption and fluorescence maxima of 492 and 535 nm. The latter values, as well as the shape of both the fluorescence and absorption curves of the second sample were superimposable on a recrystallized sample of acridine orange labeled correctly C. I. 46905. Euchrysine has two free amino groups which are fully methylated in acridine orange, therefore a nitrous acid test can differentiate the two dyes. The sample of euchrysine labeled GGNX gave a reaction, as did acridine yellow, C. I. 46025, but acridine orange, C. I. 46005, did not. Fluorescence metachromasy of euchrysine is less efficient than that of acridine orange in two ways: the shift in the spectrum is smaller by about 40 nm, making the separation of the colors more difficult both visually and by instruments and the metachromatic fluorescence has less than half of the intensity of acridine orange as measured at the peak for each dye. Confusion between these two dyes has occurred because suppliers have used the names interchangeably. For critical studies, the dye used should be identified by its Colour Index number.     

Evidence for two spectroscopically different dimers of light-harvesting complex I from green plants

A preparation consisting of isolated dimeric peripheral antenna complexes from green plant photosystem I (light-harvesting complex I or LHCI) has been characterized by means of (polarized) steady-state absorption and fluorescence spectroscopy at low temperatures. We show that this preparation can be described reasonably well by a mixture of two types of dimers. In the first dimer about 10% of all Q(y)() absorption of the chlorophylls arises from two chlorophylls with absorption and emission maxima at about 711 and 733 nm, respectively, whereas in the second about 10% of the absorption arises from two chlorophylls with absorption and emission maxima at about 693 and 702 nm, respectively. The remaining chlorophylls show spectroscopic properties comparable to those of the related peripheral antenna complexes of photosystem II. We attribute the first dimer to a heterodimer of the Lhca1 and Lhca4 proteins and the second to a hetero- or homodimer of the Lhca2 and/or Lhca3 proteins. We suggest that the chlorophylls responsible for the 733 nm emission (F-730) and 702 nm emission (F-702) are excitonically coupled dimers and that F-730 originates from one of the strongest coupled pair of chlorophylls observed in nature.     

Fluorescent photobleaching of endogenous protoporphyrin IX in yeast cells

Yeast Saccharomyces cerevisiae accumulate in the presence of chelator 2,2'-dipyridyl, two fractions of photosensitizer protoporphyrin IX, which fluoresce with maxima at 625 and 635 nm. The two fractions were found to differ from each other in the dynamics and character of fluorescence photobleaching. In contrast to the protoporphyrin IX that fluoresces with a maximum at 625 nm, the protoporphyrin IX fraction that fluoresces with a maximum at 635 nm is more photolable; in addition, upon photobleaching of its fluorescence, a new maximum at 675 nm appears.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480–530 nm and fluorescence maxima at 500–550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled oligonucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Boradiazaindacene dyes for technology of biological microchips

A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480-530 nm and fluorescence maxima at 500-550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled olgionucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.     

Di(1,N6-ethenoadenosine)5', 5'-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Di(1,N6-ethenoadenosine)5',5'-P1,P4-tetraphosphate, epsilon-(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2-chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristics maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (lambda exc 307 nm, lambda em 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split epsilon(Ap4A) and catabolize the resulting epsilon-nucleotide moieties up to epsilon-Ado.     

Interaction of hemopexin with water-soluble porphyrins.

Polyacrylamide-gel electrophoresis and filtration on Bio-Gel P-10 indicate that rabbit hemopexin binds deuteroporphyrin and 2,4-disulfonic acid deuteroporphyrin (dsDp) but not ethylenediamine-substituted protoporphyrin. Formation of the dsDp-hemopexin complex, produces a red shift in Soret maxima from 402 to 426 nm. Concomitant with this shift, the four-banded spectrum in the visible region changes to one with two absorption maxima at 554 and 590 nm. Fluorescence quenching data indicate the formation of an equimolar complex of hemopexin and dsDp with apparent dissociation constant of 1.8 × 10^?6m. The fluorescence emission maxima are at 623 nm for dsDp, at 603 nm for dsDp di-cation and at 590 nm for dsDp-hemopexin. The spectral changes following the interaction of dsDp with hemopexin may be interpreted to indicate that the porphyrin is in a less polar environment with an altered symmetry of the porphyrin nucleus. Since the dsDp-hemopexin spectrum resembles that of the di-cation we suggest that the pyrrole nitrogens of dsDp are protonated by or hydrogen bonded to two amino acids in the heme-binding site of hemopexin.     

发菜藻胆体分离和光谱特性的研究

完整藻胆体和不完整藻胆体的吸收峰都在618nm。完整藻胆体的室温荧光峰位于670nm 以上,而不完整藻胆体则在670nm以下。完整藻胆体的77K荧光发射光谱中只有648nm一个荧光发射带;而在不完整藻胆体,则有2个或3个发射带,它们位于684nm,666nm和648nm, 依次属于别藻蓝蛋白 — B,别藻蓝蛋白和C — 藻蓝蛋白的荧光。     

Picosecond and microsecond pulse laser studies of exciton quenching and exciton distribution in spinach chloroplasts at low temperatures

Studies of the fluorescence quantum yield and decay times, determined at the emission maxima of 685 and 735 nm, using picosecond laser pulses for excitation, indicate that the pigments which are responsible for the 735 nm emission derive their energy by transfer of singlet excitons from the light-harvesting pigments and not by direct absorption of photons. Microsecond pulse laser studies of the fluorescence quantum yields at these two fluorescence wavelengths indicate that long lived quenchers (most probably triplet states), which quench singlet excitons, accumulate preferentially within the long wavelength pigment system which gives rise to the 735 nm emission band.     

发菜藻胆体的分离和光谱特性的研究

完整藻胆体和不完整藻胆体的吸收峰都在618nm。完整藻胆体的室温荧光峰位于670nm以上,而不完整藻胆体则在670nm以下。完整藻胆体的77K荧光发射光谱中只有648nm一个荧光发射带;而在不完整藻胆体,则有2个或3个发射带,它们位于684nm,666nm和648nm,依次属于别藻蓝蛋白-B,别藻蓝蛋白和C-藻蓝蛋白的荧光。