Distribution of porphyrin sensitizers among protein and cellular blood elements

The distribution of porphyrin pigments between plasma proteins and blood cells was studied. It was shown that the relative fraction of sensitizer bound by blood cells changed significantly depending on the physicochemical features of pigment molecules. This parameter strongly correlates with porphyrin polarity. Polar watersoluble tetraphenylporphin derivatives, chlorine e6 and hematoporphyrin are bound by plasma proteins only. The decrease in pigment polarity by substitution of polar side groups results in a drastic increase of pigment affinity to blood cells. The binding of extremely apolar pigments by cells in blood occurs for a long period of time, probably as a result of a low rate of pigment redistribution between serum proteins and cellular membrane. The data obtained show that blood cells may be involved into the control of pigment transport and distribution in organism during photodynamic therapy. The parameters of porphyrin distribution between plasma proteins and cells in blood are of certain importance when the pharmacokinetic behavior of various sensitizers is compared.     

Selectivity of accumulation of chlorine e6 derivatives in blood leukocytes

The uptake of chlorine e6 derivatives by peripheral blood leukocytes was studied using a fluorescence-activated sorter. The analysis showed that the order of pigment uptake by leukocyte populations is the following: granulocytes > or = monocytes > lymphocytes. It was found that the accumulation of the pigments in the cell significantly varied. The level of chlorine e6 dimethyl ester accumulated by cells was found to be 15 times higher than that of chlorine e6. It was assumed that the differences in pigment uptake by different types of blood cells are due to structural and morphological features of leukocytes. The data obtained may be useful in developing the new methods of photodynamic therapy.     

Photosensitization by chlorine and porphyrin causing structural damage of erythrocytes

Kinetics of hemolysis of human erythrocytes photosensitized by porphyrin and chlorin derivatives was investigated by small angle light scattering method. The compounds used were arranged in the following order of photosensitizing activity decrease: ethylendiamide of chlorine e6, chlorine e6 dimethylester chlorine e6 haematoporphyrin diacetate tetracarboxyphenylporphyn. The substances having a greater number of binding sites with liposomes (chlorine e6 dimethylester) or with albumin (ethylendiamide of chlorine e6) showed the greatest photohemolytic activity. The photohemolytic activity of porphyrins was suggested to depend on the number of the pigment molecules bound by erythrocytes membranes.     

Photodynamic effects of a concanavalin A-chlorin e6 conjugate on human fibroblasts

To minimize the side effect of porphyrin photosensitizers and to reduce their active concentration, chlorine e6 was conjugated with concanavalin A. Photodynamic action of chlorine e6 and concanavalin A-chlorine e6 conjugate has been studied in human skin embryonic fibroblasts. The conjugate appeared to be 5 times more effective as compared to chlorine e6 due to concanavalin A-chlorine e6 endocytosis into intracellular compartments.     

Pigments of Rlepharisma zindulans Compared with Hypericin

SYNOPSIS. An analysis of the pigments from the protozoan Blepharismn undtulans is reported. Methods of isolation and separation by paper chromatography are described. along with subsequent spectrophotometric and chemical analysis. Since the two major pigments were available only in amounts insufficient for thorough chemical analysis, these pigments were compared with the known pigment hypericin. Although similarities in the visible spectra indicate that the B. unduluns pigments possess the same mesodianthrone structure as hypericin, infrared spectra and chemical reactions disclose differences in the position, and possibly in the number, of the functional groups. A tentative structure is proposed.     

Isolation and identification of the urinary pigment uroerythrin.

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative. Urine was applied to a column of Amberlite XAD-2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH-20 and thin-layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silical gel thin-layer plates. The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin-layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.     

On the evolution of the photosynthetic pigments.

During the course of terrestrial evolution, some organisms developed the capability of capturing and utilizing solar radiation. Colored compounds were undoubtedly incorporated within living forms from the earliest times, but during the transition from heterotrophic to a photoautotrophic metabolism only those pigments were selected that were components of the evolving photosynthetic apparatus and were able to catalyze reactions involving storage of light energy in chemical bonds. In this communication, some properties of tetrapyrroles with a closed porphyrin ring containing a metal ion in the center are discussed. These compounds are present in all principal contemporary photosynthetic pigments, and their synthesis has been demonstrated from simpler compounds under prebiotic conditions. It is probable that during intermediate stages in the evolution of photosynthesis, pigments with oxidizing potentials lower than that of chlorophyll were utilized to store light energy although they were not capable of removing electrons from water. The evolution and function of multiple forms of a given photosynthetic pigment in vivo are discussed. 'Accessory' pigments may be regarded as rudiments of the evolutionary development of the photosynthetic apparatus.     

FRACTIONATION OF THE EYE PIGMENTS OF DROSOPHILA MELANOGASTER

Eye pigments of normal and mutant types of D. melanogaster have been extracted with water and fractionated by chromatographic adsorption on powdered talc. Spectra of all the fractions obtainable in solution have been measured and the general chemical behavior of the pigments is described. Two chemically distinct groups of pigments are found, to be identified with the earlier designated red and brown components. The red component in the wild-type eye contains three well defined pigments, two of them capable of further subdivision so that the total number of fractions obtained is five. There is also present a brown component pigment which could not be treated quantitatively by the methods employed. All members of the wild-type red component are found in cinnabar eyes, unaccompanied by the brown component. Conversely, brown eyes contain a pigment indistinguishable from the wild-type brown component, virtually alone. In sepia eyes, one red component and two brown component pigments can be distinguished, all three pigments differing from those of wild-type eyes. Pigments apparently identical with those found in wild-type melanogaster eyes have also been found in D. virilis.     

Lipofuscin and lipofuscin-like substances

Lipofuscin is defined as being a yellowish brown, lipid-rich, heterogeneous, cytoplasmic granular pigment emitting an intense yellow autofluorescence when excited with ultraviolet light, which accumulates in various tissues of animals during their aging. It is believed that the pigments are derived from the reaction of some of reactive secondary products including malonaldehyde, formed during membranous lipid peroxidation, with amino groups of phospholipids and proteins, etc., and that these formations are accompanied by alteration of the membrane structure and inactivation of the enzymes. The fluorescence measurement of the pigments is widely used as a parameter of lipid peroxidation in vivo as well as in vitro. However, their origin, chemical structure, biological significance or fate has not as yet been fully elucidated. This article introduces and discusses the recent studies on these problems.     

MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF GOLDFISH ERYTHROPHORES AND THEIR PTERINOSOMES

The fine structure of integumental erythrophores and the intracellular location of pteridine and carotenoid pigments in adult goldfish, Carassius auratus, were studied by means of cytochemistry, paper and thin-layer chromatography, ionophoresis, density-gradient centrifugal fractionation, and electron microscopy. The ultrastructure of erythrophores is characterized by large numbers of somewhat ellipsoidal pigment granules and a well-developed system of tubules which resembles endoplasmic reticulum. The combined morphological and biochemical approaches show that pteridine pigments of erythrophores are located characteristically in pigment granules and are the primary yellow pigments of these organelles. Accordingly, this organelle is considered to be the "pterinosome" which was originally found in swordtail erythrophores. Major pteridines obtainable from goldfish pterinosomes are sepiapterin, 7-hydroxybiopterin, isoxanthopterin, and 6-carboxyisoxanthopterin. Density-gradient fractions indicate that carotenoids are mostly associated with the endoplasmic reticulum. Both tyrosinase and possibly a tyrosinase inhibitor containing sulfhydryl groups are present in the pterinosome. The possible existence of a tyrosinase inhibitor is suggested by the marked increase of tyrosinase activity upon the addition of iodoacetamide or p-chloromercuribenzoic acid. In the light of their fine structure, pigmentary composition, and enzymatic properties, the erythrophores and pterinosomes are discussed with respect to their probable functions and their relationship to melanophores.     

Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment

In the course of our studies on the structure/function relationship of visual pigments, we have expressed the human green cone pigment in the baculovirus/insect cell expression system. Purification of the human green cone pigment, however, has so far proven to be severely hampered by the low thermal stability of this receptor in a solubilized state. In order to overcome this problem, we tested a variety of chemical compounds that have been described to improve protein stability in various applications. The presence of glycerol, sucrose, trehalose and lipids during extraction improved the thermal stability of the recombinant green cone pigment up to twofold. We also analyzed the effect of mutation of residues Met208, Cys212 and Cys273 into Phe in all combinations. These mutants were designed in an attempt to increase the thermal stability by replacing weakly interacting side chains in the green pigment with their counterparts in rhodopsin with strong aromatic stacking interaction. All mutants produced wild-type levels of functional pigment, but none showed an increase in thermal stability.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super‐molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.     

Comparative studies on the late bleaching processes of four kinds of cone visual pigments and rod visual pigment

Visual pigments in rod and cone photoreceptor cells of vertebrate retinas are highly diversified photoreceptive proteins that consist of a protein moiety opsin and a light-absorbing chromophore 11-cis-retinal. There are four types of cone visual pigments and a single type of rod visual pigment. The reaction process of the rod visual pigment, rhodopsin, has been extensively investigated, whereas there have been few studies of cone visual pigments. Here we comprehensively investigated the reaction processes of cone visual pigments on a time scale of milliseconds to minutes, using flash photolysis equipment optimized for cone visual pigment photochemistry. We used chicken violet (L-group), chicken blue (M1-group), chicken green (M2-group), and monkey green (L-group) visual pigments as representatives of the respective groups of the phylogenetic tree of cone pigments. The S, M1, and M2 pigments showed the formation of a pH-dependent mixture of meta intermediates, similar to that formed from rhodopsin. Although monkey green (L-group) also formed a mixture of meta intermediates, pH dependency of meta intermediates was not observed. However, meta intermediates of monkey green became pH dependent when the chloride ion bound to the monkey green was replaced with a nitrate ion. These results strongly suggest that rhodopsin and S, M1, and M2 cone visual pigments share a molecular mechanism for activation, whereas the L-group pigment may have a special reaction mechanism involving the chloride-binding site.     

Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy

The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.     

Contrasting Patterns of Phytoplankton Community Pigment Composition in Two Salt Marsh Estuaries in Southeastern United States

Phytoplankton community pigment composition and water quality were measured seasonally along salinity gradients in two minimally urbanized salt marsh estuaries in South Carolina in order to examine their spatial and temporal distributions. The North Inlet estuary has a relatively small watershed with minimal fresh water input, while the Ashepoo, Combahee, and Edisto (ACE) Basin is characterized by a relatively greater influence of riverine drainage. Sampling stations were located in regions of the estuaries experiencing frequent diurnal tidal mixing and had similar salinity and temperature regimens. Phytoplankton community pigment composition was assessed by using high-performance liquid chromatography (HPLC) and multivariate statistical analyses. Shannon diversity index, principal-component, and cluster analyses revealed that phytoplankton community pigments in both estuaries were seasonally variable, with similar diversities but different compositions. The temporal pigment patterns indicated that there was a relatively weak correlation between the pigments in ACE Basin and the relative persistence of photopigment groups in North Inlet. The differences were presumably a consequence of the unpredictability and relatively greater influence of river discharge in the ACE Basin, in contrast to the greater environmental predictability of the more tidally influenced North Inlet. Furthermore, the timing, magnitude, and pigment composition of the annual phytoplankton bloom were different in the two estuaries. The bloom properties in North Inlet reflected the predominance of autochthonous ecological control (e.g., regenerated nutrients, grazing), and those in ACE Basin suggested that there was greater influence of allochthonous environmental factors (e.g., nutrient loading, changes in turbidity). These interestuarine differences in phytoplankton community structure and control provide insight into the organization of phytoplankton in estuaries.     

Contrasting patterns of phytoplankton community pigment composition in two salt marsh estuaries in southeastern United States

Phytoplankton community pigment composition and water quality were measured seasonally along salinity gradients in two minimally urbanized salt marsh estuaries in South Carolina in order to examine their spatial and temporal distributions. The North Inlet estuary has a relatively small watershed with minimal fresh water input, while the Ashepoo, Combahee, and Edisto (ACE) Basin is characterized by a relatively greater influence of riverine drainage. Sampling stations were located in regions of the estuaries experiencing frequent diurnal tidal mixing and had similar salinity and temperature regimens. Phytoplankton community pigment composition was assessed by using high-performance liquid chromatography (HPLC) and multivariate statistical analyses. Shannon diversity index, principal-component, and cluster analyses revealed that phytoplankton community pigments in both estuaries were seasonally variable, with similar diversities but different compositions. The temporal pigment patterns indicated that there was a relatively weak correlation between the pigments in ACE Basin and the relative persistence of photopigment groups in North Inlet. The differences were presumably a consequence of the unpredictability and relatively greater influence of river discharge in the ACE Basin, in contrast to the greater environmental predictability of the more tidally influenced North Inlet. Furthermore, the timing, magnitude, and pigment composition of the annual phytoplankton bloom were different in the two estuaries. The bloom properties in North Inlet reflected the predominance of autochthonous ecological control (e.g., regenerated nutrients, grazing), and those in ACE Basin suggested that there was greater influence of allochthonous environmental factors (e.g., nutrient loading, changes in turbidity). These interestuarine differences in phytoplankton community structure and control provide insight into the organization of phytoplankton in estuaries.     

Observations on the pigmentation of the pigeon iris

There are three genetically controlled iris types found in the pigeon, two of which contain stromal pigment cells, the third lacks pigment cells. The yellow (gravel) and white (pearl) iris types have pigment cells that contain birefringent pigment granules (crystals) and are ultrastructurally similar to iridophores of poikilothermic vertebrates. Both these iris types contain guanine as a major "pigment" and, in addition, the yellow iris contains at least two yellow fluorescing pigments that are tentatively identified as pteridines. The pigment cells of the yellow and white irises are structurally identical differing only in the presence or absence of these yellow pigments. The stromal pigment cells of the white iris correspond in structure and pigment chemistry to classical iridophores although they lack strong irridescence and are therefore perhaps best considered leucophores. The pigment cells of the yellow iris can be considered "reflecting xanthophores" having the combined properties of both classical xanthophores and iridophore/leucophores.     

The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture.

Thirty-nine strains from the genus Monascus were cultivated aerobically to study the relation between nitrogen nutrition and sporulation and pigment production. The effects of yeast extract, nitrate, ammonium, and ammonium nitrate have been compared. During cultivation the pHs of the different media are not the same, resulting in the formation of different coloured pigments. When the source of nitrogen is yeast extract or nitrate the pH is around 6.5 and red pigments are formed, whereas with ammonium or ammonium nitrate the pH is around 2.5 and the pigments are orange. It is proposed that only the orange pigments, monascorubrin and rubropunctatin, are produced biosynthetically and that the other pigments are formed from these by chemical transformations depending on the cultural conditions. The presence of organic nitrogen is optimal for growth and unfavourable for pigment production. Reduced growth and best pigment formation occurs with the three other nitrogen sources. Nitrate stimulates conidiation and sexual reproduction, while ammonium is inhibitory. Pigment production is better when conidiation is reduced. A mechanism is proposed for the control of sporulation and pigment production.     

Anthraquinone pigments from Phoma foveata Foister

1. Suitable conditions have been determined for growing the fungus Phoma foveata in culture for optimum production of pigments. 2. The pigments have been extracted and fractionated according to their solubility in aqueous bicarbonate, carbonate and alkali. 3. The carbonate- and alkali-soluble pigments have been further separated by chromatography and shown to consist of anthraquinone derivatives. 4. The alkali-soluble fraction, which comprised the bulk of the pigment, consists of pachybasin (1-hydroxy-3-methylanthraquinone; II) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone; III) by comparison with authentic samples. 5. The carbonate-soluble fraction contains emodin (1,6,8-trihydroxy-3-methylanthraquinone; V) and a hitherto unrecorded pigment for which the name phomarin and the structure 1,6-dihydroxy-3-methylanthraquinone (IV) are put forward. 6. The identification of emodin and the structural determination of phomarin are based largely on their ultraviolet, visible, infrared, nuclear-magnetic-resonance and mass spectra.