Quantitative Estimation of Coproporphyrin III and Protoporphyrin IX from Their Mixture by Spectrofluorometry

For quantitative estimation of coproporphyrin III and protoporphyrin IX from their mixture, a sensitive spectrofluorometric method was developed. At room temperature, coproporphyrin III fluoresces in neutral or alkaline pH at 622 nm having substantial fluorescence at 632 nm where protoporphyrin IX also fluoresces maximally. Similarly, protoporphyrin IX also has substantial fluorescence at 622 nm. Therefore, while estimating protoporphyrin IX (E400 F632) or coproporphyrin III (E400 F622) concentratton, it is essential to correct for the fluorescence due to coproporphyrin III at 632 nm and protoporphyrin IX at 622 nm. This was done by formulating equations from appropriate constants derived from pure samples of coproporphyrin III and protoporphyrin IX. As law as 1 pmole of coproporphyrin III or protoporphyrin IX could be estimated from their mixture by using the spectrofluorometric method.     

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.     

Protochlorophyll forms in roots of dark-grown plants

Protochlorophyll was found in roots of dark-grown plants of seven species investigated. It was identified by absorbance and fluorescence spectra of acetone and ether extracts. Chlorophyll was also found in roots of one pea species. The concentration of protochlorophyll was usually highest in young root tips and decreased upwards along the roots. The maxima of the in vivo absorbance spectra of the species studied varied between 634 and 638 nm. Low temperature in vivo fluorescence emission spectra had two maxima, one at ca 633 and the other at ca 642 nm, when the wavelengths of the excitation light were 440 and 460 nm, respectively. In vivo fluorescence excitation spectra displayed a shift of the excitation maximum from 438 to 445 nm, when emission varied from 620 to 647.5 nm. Deconvolution of these three types of spectra into Gaussian components made it possible to identify two spectral forms of protochlorophyll: protochlorophyll_629–633 and protochlorophyll_638–642.     

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.     

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.     

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 °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 °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 °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 °C.     

Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus

Chlorophyll precursors Mg-protoporphyrin IX and its monomethylester are candidates for plastid-derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+-containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs-1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light-induced increase in the levels of Mg2+-containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light-induced rise in pool levels and induction of the HSP70 genes. Similarly, pre-gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor-treated cells and in pre-gametes could be circumvented by the exogenous addition of Mg-protoporphyrin IX in the dark. This suggests an essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway. However, accumulation of this porphyrin in the dark - presumably in the chloroplast - did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.     

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.     

Fluorescence properties of melanins from opioid peptides.

Recently our group synthesized a new class of melanins obtained by the tyrosinase-catalyzed oxidation of opioid peptides (opiomelanins). Owing to the presence of the peptide moiety such pigments exhibit high solubility in hydrophilic solvents, which allows spectroscopic investigations. In particular, the absence of solid-state quenching effects enables the study of melanin fluorescence properties, till now poorly investigated due to the complete insolubility of melanins produced from tyrosine or Dopa. Opiomelanins dissolved in aqueous medium show a characteristic emission peaked at 440 and 520 nm when excited around 330 nm, where a maximum is observed in the absorption spectrum. Kinetic measurements performed on the tyrosinase-catalyzed oxidation of opioid peptides show that the 440-nm fluorescence band arises in the early stages of peptide oxidation, whereas the 520-nm band appears in later stages of oxidation, i.e., during the polymerization of indole-quinone units. Moreover, molecular sieve fractionation shows that in the opiomelanin fraction with a molecular weight lower than 10 kDa the 440-nm band is dominant in the fluorescence spectrum. The breakdown of the polymer induced by hydrogen peroxide and light (i.e., the photobleaching of melanin pigments) produces a marked enhancement of the 440-nm fluorescence band while the 520-nm band disappears. Hence, our findings suggest that the observed fluorescence contains contributions from both oligomeric units (440-nm band) and high-molecular-weight polymers (520-nm band).     

Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.     

Native state,energetic interaction of chlorophyll precursors and intraplastid location of S-adenosyl-L-methionine: Mg-protoporphyrin IX methyltransferase in etiolated leaves

Low temperature fluorescence spectra (FS) and fluorescence excitation spectra (FES) of protoporphyrin IX (Proto), Mg-protoporphyrin IX and its monomethyl ester (MgProto-ME) and protochlorophyllide (Pchlide) in etiolated barley leaves treated with 5-aminolevulinic acid and/or 2,2'-dipyridyl were studied. The spectra of Proto and MgProto-ME showed a little dependence on temperature of registration and exhibited similarity to low temperature spectra in diluted organic and buffer solutions. However, a red wavelength shift for Soret bands of Proto and MgProto-ME was observed due to porphyrin interaction with bovine serum albumin in 0.05 M, Na2HPO4 solution at room temperature. Disaggregating treatments had no effect on Proto and MgProto-ME spectra in plants. These results suggested that in etiolated leaves Proto and MgProto-ME molecules were in a monomer state. The spectral properties of these molecules were determined by interaction of porphyrins with proteins and other plastid membrane components. The spectral analyses indicated an efficient energy migration from Proto and MgProto-ME molecules to active form of Pchlide which emitted at 656nm, and no energy transfer from carotenoids to porphyrins in vivo. These findings suggested that Proto and MgProto-ME from carotenoids, and close location of these porphyrins and photoactive Pchlide in etioplast membranes. The latter conclusion was strongly supported by an observation that in etiolated leaves, S-adenosyl-L-methionin:Mg-protoporphyrin IX methyltransferase, which converts MgProto into MgProtoME, were located not only in prothylakoids but also in prolamellar bodies containing photoactive Pchlide.     

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.     

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.     

Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths

Indo-1 is a new fluorescent indicator of the intracellular free calcium concentration Cai++. Indo-1 may be used in a similar manner as its predecessor quin2 but offers the principal advantage that the Ca++ saturated form of the Ca++ chelator has a emission maximum different in wavelength from that of free indo-1 (400 nm versus 483 nm). Therefore, the ratio of the fluorescence intensity F emitted at 400 nm to that of the fluorescence intensity G emitted at 483 nm (or 500 nm) should be a measure of Cai++ independent of the total amount of intracellular dye. However, when indo-1 is loaded into endothelial cells (grown in culture on quartz coverslips) by incubation with the acetoxymethylester of indo-1 (indo-1/AM), the ester in not completely hydrolysed to indo-1 intracellularly. Fluorescence emitted by uncleaved indo-1/AM at wavelengths 483-500 nm interferes with the fluorescence of indo-1. Ester fluorescence is influenced not only by ester concentration but by the fluorescence emitted at 400 nm by Ca++ bound indo-1 as well. Therefore, the ratio F/G cannot reliably evaluate increases in Cai++ in endothelial cells although F/G would indicate a basal Cai++ constant with time. By contrast, the fluorescence F is a sensitive parameter of the intracellular concentration of Ca++ bound indo-1, in particular when the excitation wavelength is set to 332 nm. F was used to measure resting Cai++ in endothelial cells (132 +/- 22 nM; n = 22) and to demonstrate dose-dependent and reversible increases in Cai++ in response to stimulation with bradykinin.     

Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.     

Separation of Mg-Protoporphyrin IX and Mg-Protoporphyrin IX Monomethyl Ester Synthesized de novo by Developing Cucumber Etioplasts

High pressure liquid chromatography was used to demonstrate that chelation of Mg²⁺ into protoporphyrin IX precedes methylation in isolated greening etioplasts from cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. Mg-protoporphyrin IX synthesized in vitro from protoporphyrin IX, Mg²⁺, and ATP or exogenous Mg-protoporphyrin IX could serve as substrates for the methylation step. In either case, S-adenosylmethionine was the methyl donor and could not be replaced by ATP plus methionine.     

Differential distribution of chlorophyll biosynthetic intermediates in stroma, envelope and thylakoid membranes in Beta vulgaris

Stroma, envelope and thylakoid membranes were prepared from chloroplasts isolated from leaves of Beta vulgaris. Out of total plastidic protochlorophyllide, envelope membranes contained 1.5%, thylakoids had the maximum 98.48% and stroma had a trace fraction of 0.02%. Distribution of the Mg-protoporphyrin IX and its monoester was 89.0% in thylakoids, 10.0% in stroma and 1.0% in envelope. A substantial fraction (33.77%) of plastidic protoporphyrin IX was partitioned into stroma. Envelope contained 0.66% and thylakoids had 65.57% of the total plastidic protoporphyrin IX pool. The proportion of monovinyl and divinyl forms of protochlorophyllide was almost similar in intact plastid, thylakoids, and outer and inner envelope membranes suggesting a tight regulation of vinyl reductase enzyme. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma is discussed. This work was supported by a grant from the Council of Scientific and Industrial Research (38/1079/03/EMRII) to BCT.     

Bovine serum albumin-(7-hydroxycoumarin-4-acetic acid) complex: applications to the fluorometric measurement of fatty acid concentrations

A covalent complex between bovine serum albumin and 7-hydroxycoumarin-4-acetic acid (BSA-HCA) shows a strong fluorescence band at lambdamax = 450 nm upon excitation at 375 nm. Quenching of the fluorescence emission accompanies the association of fatty acids (FA) to BSA-HCA and the application of the complex as a spectrofluorometric probe for measurement of fatty acid concentrations in aqueous solution is examined. Binding constants for various long-chain fatty acids (Kd = 14-460 nM) and calibration curves characterizing the probe have been determined. Standardized assay conditions allow for accurate measurements in the concentration range of 10 nM to 5 microM. BSA-HCA provides a stable and sensitive fluorescence-based FA probe with potential biochemical applications.     

The physicochemical state of protoporphyrin IX in aqueous solution investigated by fluorescence and light scattering

Fluorescence spectros copy and light scattering have been used to investigate the physicochemical behaviour of protoporphyrin IX in aqueous solutions. In the alkaline range large micelles are formed with a hydrodynamic radius of 130 nm and a molecular mass of 5.0 x 10(7) Da. The micelles are fluorescent with an emission maximum at 620 nm. A pH lowering caused quenching of the micelle fluorescence. On a collision encounter these micelles will disintegrate and they are reformed by nucleation of collision fragments. From measurements of the fluorescence intensity of the micelles versus total concentration an equilibrium constant of 4.0 x 10(6) M(-1) was found for this collision-nucleation process. In the pH range between 6 and 3 another micelle type of twice the size of those in the alkaline range was stable with respect to the solute. These micelles have free base porphyrin fluorescence with an emission maximum at 634 nm. A lowering of the pH below unity causes disintegration of these micelles and monomer fluorescence from the protoporphyrin dication was observed.     

A picosecond pulse train study of exciton dynamics in photosynthetic membranes.

The fluorescence decay time of spinach chloroplasts at 77 degrees K was determined at 735 nm (corresponding to the photosystem I emission) using a train of 10-ps laser pulses spaced 10 ns apart. The fluorescence lifetime is constant at congruent to 1.5 ns for up to the fourth pulse, but then decreases with increasing pulse number within the pulse train. This quenching is attributed to triplet excited states, and it is concluded that triplet excitons exhibit a time lag of about 50 ns in diffusing from light harvesting antenna pigments to photosystem I pigments. The diffusion coefficient of triplet excitons is a least 300--400 times slower than the diffusion coefficient of singlet excitons in chloroplast membranes.