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We have shown earlier that yeast cells grown in synthetic mediums supplemented with Zn++ accumulate large amounts of Zn-protoporphyrin within their mitochondria. This accumulation is accompanied by an inhibition of respiration (3). This study deals with the effect of light on the respiratory inhibition and the release of respiratory control which are observed if Zn-protoporphyrin is added to isolated mitochondria which are initially devoid of this pigment. In addition, we have studied the effect of light on the respiratory inhibition exerted by Zn-protoporphyrin accumulated in vivo. The following results were obtained: 1) The light-induced destruction of Zn-protoporphrin which had been added in vitro to Zn-protoporphyrin-free mitochondria significantly inhibits respiration and phosphorylation. Under these conditions, the extent of the inhibitions increases with the concentration of the added Zn-protoporphyrin and the duration of illumination. 2) Accumulation of Zn-protoporphyrin within the cells causes an inhibition of the respiratory activities and the activities of succinate-cytochrome c reductase and NADH-cytochrome c reductase of the mitochondria. Illumination of the isolated mitochondria from Zn-protoporphyrin-containing cells enhances the inhibition of these activities. No light-induced inhibition of these activities is observed with mitochondria from cells devoid of Zn-protoporphyrin.
We describe the separation of protoporphyrin and related porphyrins by reversed-phase "high-performance" liquid chromatography, with fluorometric detection. We used the method to demonstrate that acid hydrolysis of the dimethyl ester of protoporphyrin IX is complete in 2 to 3 h and is followed by the acid-catalyzed conversion of protoporphyrin IX to a chemical species that chromatographic evidence indicates to be hematoporphyrin IX. In addition, the method was used to evaluate the purity of a commercial preparation of protoporphyrin IX and was also demonstrated to have the sensitivity and specificity needed for measuring the protoporphyrin IX content of whole-blood.
The effects of ultraviolet and visible irradiation in presence of protoporhyrin upon mast cells were investigated. Rat peritoneal mast cells were suspended in a medium containing varying concentrations of protoporphyrin and were irradiated using a mercury vapor lamp. The release of histamine is related to both the concentrations of protoporphyrin and the period of irradiation. Comparatively negligible amounts of histamine were released when the cells were irradiated in absence of protophyrin or when the cells were incubated in the dark in presence of protoporphyrin. When 51Cr-labelled cells were employed, the release of histamine was paralleled by the release of 51Cr. These results suggest that the release of histamine was due to the lysis of cells. It was found that 2-deoxyglucose, iodoacetate and carbonyl cyanide, m-chlorophenyl hydrazone at concentrations which are known to inhibit the secretory release of histamine or uncouple oxidative phosphorylation, had no effect on the photolysis of mast cells. When mast cells were irradiated in presence of protoporphyrin for 5 to 10 minutes and then kept in the dark, the release of histamine was greater than when the cells were irradiated but not subsequently kept in the dark. It is suggested that the initial site of photochemical lysis is at or near the cell surface.
1. Treatment of rats with small doses of CoCl2 decreases liver 5-aminolaevulinate synthase (EC 2.3.1.37) activity and impairs incorporation of 5-amino[14C]laevulinate into liver haem. Salts of other metals (cadmium, nickel, manganese and zinc) are all relatively inactive. 2. The dose-response curves obtained for both these effects closely mirror the accumulation in the liver of a compound that is labelled by 5-amino[14C]laevulinate and is unextractable by acetone/HCl. 3. Incorporation of 5-amino[14C]laevulinate into unextractable compound is also obtained in vitro by incubating liver homogenates with label in the presence of cobalt:isotope-dilution experiments show that the radioactivity passes through pools of porphobilinogen and protoporphyrin, but not of haem. 4. The unextractable compound is not covalently bound to protein and possesses the same extraction and spectral properties as authentic cobalt protoporphyrin. 5. It is concluded (a) that cobalt protoporphyrin is readily formed not only in vitro, but also in vivo, and (b) that its formation accounts for the impaired incorporation of 5-aminolaevulinate into haem and may also be responsible for the action of cobalt on 5-aminolaevulinate synthase.
Haemin and protoporphyrin IX, but not bilirubin, are extensively bound by human spectrin. The absorption spectrum of the bound haemin is indicative of coordination of the iron by nitrogenous ligands in the protein. The protoporphyrin IX generates difference spectra on binding, which change with ligand:protein ratio, showing the existence of at least two structurally distinct types of site. The binding of both ligands is complex, and may be cooperative. Binding isotherms, based on spectrophotometric titrations, are given. Haemin and protoporphyrin IX also bind strongly to erythrocyte ghosts. At ionic strengths near physiological we can find no evidence of binding of haemoglobin to spectrin, as judged by sedimentation velocity, and it appears that reported interactions of this nature represent only non-specific binding at low ionic strength.
The matrix fraction of rat liver mitochondria contains several components capable of binding protoheme IX and protoporphyrin IX in vitro. When binding is analyzed by sucrose density gradient centrifugation, two main groups of components can be separated; i.e. one of high and one of low molecular weight. Experimental evidence is presented indicating a protein nature of these components, and at least six species were detected by gel electrophoresis. The binding of protoheme IX and protoporphyrin IX to matrix components is also evident from a marked change in the heme and porphyrin absorbance spectra in the presence of added matrix. The data obtained indicate that the matrix fraction has a high total binding capacity for heme, but the affinity for heme of the various components seems to be different. Based on the separation of bound and free heme by gel electrophoresis it is concluded that the heme-binding capacity of the high affinity components does not exceed 14 nmol heme . mg-1 protein.
Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.
The hemin effect on protein synthesis of human granulocytes, lymphocytes and platelets was examined. Hemin added to culture media without serum caused a dose-dependent inhibition of protein synthesis in all three cell types. A cell-specific enhancement of protein-synthesizing capability was observed in 24-hour cultures in the presence of hemin and serum. A marked increase of protein synthesis was found in granulocytes, unchanged in lymphocytes and decreased in platelets. Lymphocytes from patients with chronic lymphatic leukemia (CLL) were moderately inhibited by hemin when incubated in media containing serum, the effect being more pronounced in the presence of freshly disolved doses of hemin. Addition of protoporphyrin IX to cells cultures resulted in a marked suppression of protein synthesis by the three cell types, in all experiments. These results confirm the importance of serum proteins in preventing the inhibitory effects of free hemin and protoporphyrin IX on blood cell protein synthesis. On the other hand, they show a cell-specific enhancement of the protein-synthesizing capacity mediated by hemin.
An inexpensive portable fluorometer has been designed which can be used to determine the concentration of zinc protoporphyrin (ZPP) in a drop of unprocessed blood deposited on a disposable glass slide. No volume measurement of the blood sample need be made. The instrument displays the ZPP level in micrograms per deciliter of blood accurately and reproducibly, automatically calibrating each determination by comparing the blood fluorescence with that of a stable dye. The instrument, which we call a hematofluorometer, is ideally suited for use in primary screening for childhood and occupational lead intoxication. It can be used by public health personnel after a few minutes of instruction. Comparisons of ZPP levels obtained with the use of the hematofluorometer with erythrocyte protoporphyrin levels obtained using accepted extraction methods are presented.
Inhibition of superoxide dismutase by diethyldithiocarbamate or cyanide increases the rate of red blood cells lysis after irradiation in the presence of protoporphyrin IX. Catalase activity, which is decreased during the photohemolytic process, appears to be not essential for the lytic event. No relationship between catalase activity and hemolysis rate was found. Superoxide dismutase appears to prevent only in part catalase inactivation by singlet oxygen.
Of 13 patients with Friedreich's ataxia (Type Ia) and 17 with type IIa recessive ataxias, all were found to have levels of "free erythrocyte protoporphyrin" (FEP) above the normal range. The rise in FEP in Friedreich's ataxia correlated well with the age of the individual and thus appears to be related to the course of the disease. Subjects with olivo-ponto-cerebellar atrophy, Charlevoix syndrome, Duchenne muscular dystrophy, and Parkinson's disease were also found to have significantly elevated FEP, although the distribution overlapped with the normal range. The finding of elevated FEP may indicate a relative heme deficiency in ataxia due to inhibition of ferrochelatase leading to a state of ineffective, persistent erythropoiesis. The possibility of a prostaglandin abnormality being related to this defect and to the pathogenesis of ataxia is considered.
Levels of erythrocyte delta-aminolevulinate dehydratase [ALA-dehydratase; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], UROPORPHYRINOGEN-I synthase [Uro-synthase; porphobilinogen ammonia-lyase (polymerizing), EC 4.3;1.8], AND PROTOPORPHYRIN IX (Proto) were measured by sensitive semimicroassays using 2-5 mul of whole blood obtained from normal and anemic mutant mice. The levels of erythrocyte ALA-dehydratase and Uro-synthase showed marked developmental changes and ALA-dehydratase was influenced by the Lv gene. Mice with overt hemolytic diseases (ja/ja, sph/sph, nb/nb, ha/ha) had 10- to 20-fold increases in ALA-dehydratase, Uro-synthase, and Proto compared with their normal controls. Mice with an iron deficiency (mk/mk) and mice with hypoplastic anemias (W/Wv, Sl/Sld, an/an) had mild to moderate increases in these parameters. Elevated enzyme activities and Proto correlated well with the number of reticulocytes. Because all mice with anemias possessed elevated levels of ALA-dehydratase, Uro-synthase, and Proto independent of differences in their genotypes, the increase in these parameters is not likely to be the result of a specific gene defect. The increased enzyme activities and Proto concentration probably reflect increased frequency of young red cells that are still active in heme biosynthesis.
Zinc protoporphyrin (ZnP) has been identified by several investigators as the predominant fluorescent porphyrin accumulating in erythrocytes as a result of chronic lead absorption or iron deficiency anemia. This report describes an evaluation of a new portable hematofluorometer for the determination of ZnP in finer puncture or venous blood samples. Samples were obtained from a number of employees in various job categories at steel operations throughout the United States to study the utility of ZnP as a biologic monitoring method in the occupational environment. Urinary lead, urinary aminolevulinic acid and blood lead were also determined on the same employees to examine any relationships among these measurers and to estimate a biologic threshold limit value for ZnP. The results of this investigation show that there is little elevation of the ZnP level with increasing blood level until the blood lead concentration reaches the area of 50--60 micrograms/dl. A biologic threshold level of 300 micrograms/dL, which correlates with a blood level of 60 micrograms/dL, is suggested as a guideline value for identifying workers requiring further investigation.
Soybean apoleghemoglobin a was irradiated with visible light in the presence of different sensitizers to probe the heme environment of the protein. With protoporphyrin IX as sensitizer, specific photooxidation of histidine-92 and histidine-61 occurred. Irradiation of oxyleghemoglobin and cyanleghemoglobin resulted in photooxidation of histidine-92, while addition of methylene blue caused both histidine-92 and histidine-61 to be oxidized. Apoleghemoglobin, irradiated in the presence of rose bengal or methylene blue, lost tryptophan-128 in addition to the two histidines.
In order to clarify the action of drugs for liver disease, the effect of protoporphyrin (PP) on CCl4-induced liver injury was studied. Attention was given to the levels of lysosomal enzymes, some components of the liver, and inhibition of enzymes and lysis of lysosomal membranes by lipid peroxides. Administration of PP to CCl4-poisoned rats was found to prevent the decrease in lysosomal lipolytic enzyme level in the liver, but not in other enzyme levels tested. The inhibition of lipolytic enzyme by CCl4 administered may be partially involved in lipid accumulation in the liver. A dose of PP administered to CCl4-poisoned rats for 8 days depressed the neutral lipid content in the liver nearly to the control value. Methyl linoleate hydroperoxide (hydroperoxide) at a lower concentration of 10(-6)% inhibited the lipolytic enzyme acitivity by 30% and in concentrations ranging from 10(-4) to 10(-3)% inhibited beta-glucuronidase activity. Addition of PP to the medium containing 10(-6) to 10(-5)% hydroperoxide and alpha-tocopherol reduced the enzyme inhibition further than in the absence of PP. The hydroperoxide in concentrations varying from 10(-6) to 10(-3)% caused a partial lysis of liver lysosomal membranes, but addition of PP slightly reduced the damage by the hydroperoxide in concentration lower than 10(-5)%.
A reconstituted heme oxygenase system which was composed of a purified heme oxygenase from pig spleen microsomes and a partially purified NADPH-cytochrome c reductase from pig liver microsomes could not catalyze the conversion of cobaltic protoporphyrin IX (Co-heme) to biliverdin, although Co-heme could bind with the heme oxygenase protein to form a complex. The heme oxygenase system in the microsomes from pig spleen, rat spleen, and rat kidney also failed to oxidize Co-heme to biliverdin. Properties of the complex of Co-heme and heme oxygenase closely resembled those of cobalt myoglobin and cobalt hemoglobin; the Co-heme bound to the heme oxygenase protein did not react with cyanide and azide, the Co-heme moiety was reduced but only slowly with sodium dithionite, and the reduced form of the Co-heme did not appear to bind carbon monoxide. The co-heme bound to heme oxygenase was not reduced with the NADPH-cytochrome c reductase system in air. These findings further support the views that heme oxygenase may have a heme-binding crevice similar to those of myoglobin and hemoglobin and that reduction of heme is the prerequisite for the oxidative degradation of heme in the heme oxygenase reaction.
Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.