Copper Protoporphyrin Research Articles

Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes.

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3',6'-di(O-thiosalicyl)fluorescein, SSP4), and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n = 1-7) and H2S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

Micellar Catalysis: Bioinspired Micellar Copper Protoporphyrin as a High Performing Nano-Catalyst.

Herein we have engineered a micellar Cu protoporphyrin catalyst that mediates carbon bond activation using peroxide as an electron source. Cu protoporphyrin is a biomimetic model of active site of chloroperoxidase enzyme, which catalyzes the carbon bond halogenation in the presence of a suitable amount of H2O2. The encapsulation of Cu(II) Protoporphyrin IX/L-Cysteine inside of cetyltrimethylammonium bromide micelle increases the rate of chlorination at pH 3. The cited catalyst resists high concentrations of hydrogen peroxide, which is previously reported as a suicide inactivator component of hemo-enzymes. Isothermal Titration Calorimetry (ITC) and Dynamic Light Scattering (DLS) data have revealed the formation of a micellar complex by encapsulation of six Cu(II) proporphyrins within each micelle. Moreover, electrochemical investigations indicate that L-Cysteine increases the intensity of electron transferred due to the formation of self-assembled monolayer on Au electrode. Our results paved a road toward the design of a more robust mimetic catalysis based on Protoporphyrin IX derivatives.

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

PurposeProstaglandin (PG) E2 is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE2 on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE2-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.MethodsWe explored the involvement of HO-1 on PGE2 regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.ResultsLPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE2 or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE2. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.ConclusionsAKAP plays an important role in PGE2 regulation of COX-2 expression, and the suppression of HO-1 by PGE2-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.

Metal Ion Substrate Inhibition of Ferrochelatase

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Relaxation of canine gallbladder to nerve stimulation involves adrenergic and non-adrenergic non-cholinergic mechanisms.

Electrical field stimulation (EFS) of dog gallbladder strips induced a frequency-dependent contractile response followed by an off-relaxation that was turned into a pure inhibitory response after atropine pretreatment. Guanethidine reduced the atropine-induced relaxing responses, so an adrenergic mechanism can partially account for the nerve-mediated gallbladder relaxation. However, guanethidine pretreatment also revealed a nonadrenergic noncholinergic (NANC) relaxation induced by EFS, which was frequency independent. NANC relaxations were reduced by L-arginine methyl ester (L-NAME, 100 micromol L-1), a nitric oxide synthase inhibitor (D-p-Cl-Phe6, Leul7; 10 micromol L-1), a vasoactive intestinal peptide (VIP) receptor antagonist, and an inhibitor of haem oxygenase, (copper protoporphyrin IX; CuPP-IX; 10 micromol L-1), suggesting that nitric oxide (NO), VIP and carbon monoxide (CO), respectively, are released in response to EFS. Immunoreactivities for haem oxygenase-2 (HO-2) and VIP, and histochemical staining for NADPH diaphorase were observed in nerve cell bodies and fibres, demonstrating the presence of CO, VIP and NO as putative NANC neurotransmitters in dog gallbladder. These data support the hypothesis that NO, VIP and CO contribute to NANC relaxation of the canine gallbladder.

Overexpression of heme oxygenase-1 protects allogeneic thyroid grafts from rejection in naive mice

Background. Endocrine allografts are an option for the treatment of endocrine failure. Methods. One lobe of the thyroid was transplanted under the kidney capsule. Results. C57BL/10 (H2b) thyroids were rejected in naive CBA (H2k) mice within 14 days after transplantation. When mice were treated with anti-CD4 monoclonal antibodies (mAb), all grafts survived for more than 60 days. The first grafts still survived after second C57BL/10 or Balb/c (H2d) thyroid grafts that were transplanted into the same recipients were rejected acutely, which suggests that the primary grafts were modified under anti-CD4 mAb treatment. To confirm this hypothesis, C57BL/10 thyroid grafts from anti-CD4 mAb-treated mice were retransplanted. All grafts survived in naive mice; this correlated with the overexpression of heme oxygenase-1 (HO-1) in the grafts. Next, an inhibitor of HO-1 (zinc protoporphyrin) or control compound (copper protoporphyrin) was injected intraperitoneally after transplantation of C57BL/10 thyroid grafts into the primary CBA recipients that had been treated with anti-CD4 mAb. The grafts in mice that had been treated with zinc protoporphyrin, but not copper protoporphyrin, were rejected when retransplanted to naive recipients. Conclusions. Overexpression of HO-1 correlated with the protection of fully allogeneic thyroid grafts from rejection when retransplanted into naive recipients. (Surgery 2000;128:910-7.)

Overexpression of haem oxygenase 1 protects allogeneic thyroid grafts from rejection in naive mice

Abstract Background Endocrine allografts are an option for the treatment of endocrine failure. This study examined whether anti-CD4 monoclonal antibody (mAb) could induce survival of allogeneic thyroid grafts and investigated the mechanisms involved in graft survival. Methods and Results One lobe of thyroid was transplanted under the kidney capsule and graft survival was examined histologically. C57BL/10 (H2b) thyroids were rejected in naive CBA (H2k) mice within 14 days after transplantation. When mice were treated with a depleting anti-CD4 mAb (YTA 3·1; 100 μg on day −1, 0 and 3), all C57BL/10 thyroid grafts survived for over 30 days. In order to determine if donor-specific tolerance had been induced, mice with the surviving C57BL/10 thyroid grafts were transplanted with second C57BL/10 or Balb/c (H2d) thyroids under the left kidney capsule at 30 days. Interestingly, all of the second grafts were rejected in the left kidney capsule but the first C57BL/10 thyroid grafts continued to survive in the right kidney capsule for a further 30 days. These findings suggest that grafts themselves were modified under anti-CD4 mAb treatment. To confirm this hypothesis, C57BL/10 thyroid grafts in mice treated with anti-CD4 mAb were resected and retransplanted into naive mice 30 days after transplantation. All of the grafts survived when retransplanted to naive mice and this was found to correlate with overexpression of haem oxygenase 1 in the thyroid grafts. In an attempt to demonstrate the pivotal role of haem oxygenase 1 on graft survival, an inhibitor of haem oxygenase 1 (zinc protoporphyrin) or control compound (copper protoporphyrin) that did not inhibit the enzyme was injected intraperitoneally (30 mmol l−1 every day) after transplantation of C57BL/10 thyroid grafts into the primary CBA recipients treated with anti-CD4 mAb. The grafts in mice treated with zinc protoporphyrin but not copper protoporphyrin were rejected when retransplanted to naive recipients. Conclusion Overexpression of haem oxygenase 1 protects allogeneic thyroid grafts from rejection in recipients treated with anti-CD4 mAb. This observation may be applied for clinical endocrine grafts using gene transfer of haem oxygenase 1.

Solubilization, Partial Purification, and Characterization of a Fatty Aldehyde Decarbonylase from a Higher Plant, Pisum sativum

Enzymatic decarbonylation of fatty aldehydes generates hydrocarbons. The particulate enzyme that catalyzes the decarbonylation has not been solubilized and purified from any organism but a green alga. Here we report the solubilization, purification, and partial characterization of the decarbonylase from a higher plant. Decarbonylase from a particulate preparation from pea (Pisum sativum) leaves, enriched in decarbonylase, was solubilized with β-octyl glucoside and partially purified. SDS–PAGE showed a major protein band at 67 kDa. Rabbit antibodies raised against this protein specifically cross-reacted with the 67-kDa protein in solubilized microsomal preparations; anti-ribulose bisphosphate carboxylase cross-reacted only with the 49-kDa large subunit of the carboxylase, but not with any protein near 67 kDa, showing the absence of any contamination from cross-linked small–large subunit of the carboxylase found in the green algal enzyme preparation. Anti-67-kDa protein antibodies inhibited decarbonylation catalyzed by the enzyme preparations, showing that this protein represents the decarbonylase. Decarbonylase activity of the purified enzyme required phospholipids for activity; phosphatidylcholine was the preferred lipid although phosphatidylserine and phosphatidylethanolamine could substitute less effectively. Half-maximal activity was observed at 40 μM octadecanal. The purified enzyme produced alkane and CO and was inhibited by O2, NADPH, and DTE. Metal ion chelators severely inhibited the enzyme and Cu2+ fully restored the enzyme activity. Purified enzyme preparations consistently showed the presence of Cu, and copper protoporphyrin IX catalyzed decarbonylation. These results suggest that this higher plant enzyme probably is a Cu enzyme unlike the green algal enzyme that was found to have Co.

Carbon monoxide-mediated alterations in paracellular permeability and vesicular transport in acetaminophen-treated perfused rat liver.

This study aimed to examine whether acetaminophen (AAP), an anti-inflammatory agent producing hepatocellular damages with its overdose, evokes hepatocellular dysfunction through mechanisms involving carbon monoxide (CO) generated by heme oxygenase (HO). In perfused rat livers, CO and bilirubin were determined in venous perfusate and bile samples as indices of heme degradation. Biliary excretion of transportally injected horseradish peroxidase was also determined to assess paracellular junctional permeability and vesicular transport across hepatocytes. AAP at 20 mmol/L induced a transient choleresis, followed by a reduction of bile output. Under these circumstances, the release of CO and bilirubin IXalpha, terminal products of the HO-mediated heme degradation, became 2. 5-fold greater than the control. The rate of CO production appeared stoichiometric to the degradation rate of microsomal cytochrome P-450. Mechanisms for the AAP-induced cholestasis involved an increase in the junctional permeability that coincided with a reduction of vesicular transport across hepatocytes. Clotrimazole, a cytochrome P-450 inhibitor, or zinc protoporphyrin IX, an HO inhibitor, but not copper protoporphyrin IX, which did not inhibit HO, attenuated these AAP-induced changes. Furthermore, administration of CO at concentrations comparable with those induced by AAP elicited a marked elevation of the paracellular junctional permeability concurrent with a reduction of transcellular vesicular transport, mimicking effects of the AAP administration. Thus, CO serves as a putative regulator of hepatocellular function that is overproduced through acute heme degradation during xenobiotic transformation.

The comparative effects of the NOS inhibitor, nω-nitro-l-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow

Purpose: To determine the relative effects of inhibiting nitric oxide synthase (NOS) and haemoxygenase (HO) on blood flow to the rat P22 carcinosarcoma. Methods and Materials: HO is the enzyme responsible for in vivo production of carbon monoxide (CO). The vascular effects of zinc protoporphyrin IX (ZnPP), a competitive inhibitor of HO, were compared with those of copper protoporphyrin IX (CuPP), a poor inhibitor of HO, in isolated ex vivo perfusions of the P22 tumour and in intact tumour-bearing rats. In ex vivo perfusions, tumour vascular resistance was calculated from measurements of perfusion pressure at a known flow rate. In intact animals, blood flow to tumour and normal tissues was calculated using a radiotracer uptake method. The effects of ZnPP were compared with those of the NOS inhibitor, N ω-nitro- l-arginine (L-NNA), and the combination of the two drugs. Results: HO activity in the P22 tumour was reduced by 50% following administration of either ZnPP or CuPP directly to ex vivo perfused tumours, suggesting an indirect effect on the enzyme. Enzyme inhibition was not associated with any significant vasoactive effect. Neither ZnPP nor CuPP, at a dose of 45 μmol · kg −1 administered i.p., inhibited tumour HO in vivo. However, they did significantly decrease tumour blood flow to 60–70% of control, with similar effects in skin and brain. Skeletal muscle blood flow was increased to 150% of control. L-NNA decreased both tumour and skeletal muscle blood flow to around 40% of control. These differences suggest that the nonspecific effects of ZnPP and CuPP were not mediated by NOS inhibition. The combination of ZnPP and L-NNA improved the selective reduction in tumour blood flow achieved with either agent alone. Conclusion: This suggests that the HO/CO pathway does not play a major vasodilatory role in this tumour. However, ZnPP and CuPP could be useful for inducing a relatively selective decrease in tumour blood flow via mechanisms unrelated to HO inhibition, especially when combined with NOS inhibition.

Carbon monoxide: a role in carotid body chemoreception.

Carbon monoxide (CO), produced endogenously by heme oxygenase, has been implicated as a neuronal messenger. Carotid bodies are sensory organs that regulate ventilation by responding to alterations of blood oxygen, CO2, and pH. Changes in blood gases are sensed by glomus cells in the carotid body that synapse on afferent terminals of the carotid sinus nerve that projects to respiratory-related neurons in the brainstem. Using immunocytochemistry, we demonstrate that heme oxygenase 2 is localized to glomus cells in the cat and rat carotid bodies. Physiological studies show that zinc protoporphyrin IX, a potent heme oxygenase inhibitor, markedly increases carotid body sensory activity, while copper protoporphyrin IX, which does not inhibit the enzyme, is inactive. Exogenous CO reverses the stimulatory effects of zinc protoporphyrin IX. These results suggest that glomus cells are capable of synthesizing CO and endogenous CO appears to be a physiologic regulator of carotid body sensory activity.

Collision‐activated dissociation studies of alkylamines formed from copper‐induced dealkylation of N‐alkylporphyrins

A low-energy (5-450 eV) collision-activated dissociation (CAD) study of a series of aliphatic amines revealed that at collision energies above 200 eV, charge-site-initiated fragmentation occurs. The resulting fragment ions can be utilized in the characterization of alkyl substituents of di- and trisubstituted aliphatic amines. In the presence of Cu2+ and a suitable nucleophile, such as n-dodecylamine, N-alkyl protoporphyrins dealkylate to afford copper protoporphyrin and an alkyl-dodecylamine adduct. A CAD study of a number of alkyl-dodecylamine adducts derived from the copper-induced dealkylation of synthetic N-alkyl protoporphyrins, using charge-site-initiated fragment ions, showed that the alkyl group was trapped by the nucleophilic amine present. Subsequently this method was used to identify the alkyl group of a biologically derived N-alkyl protoporphyrin.

ChemInform Abstract: STUDIES ON COPPER‐PROTOPORPHYRIN‐IRON(III) COMPLEXES. A POSSIBLE MODEL FOR CYTOCHROME C OXIDASE

AbstractDurch Zugabe von Cu‐nitratlösung in entsprechenden Mengen zu Hämatinlösungen wurden die Titelverbindungen mit einer 1:1‐ bzw. 2:1‐Cu : Fe‐ Stöchiometrie erhalten.

Studies on copper–protoporphyrin–iron(III) complexes. A possible model for cytochrome c oxidase

Methods for preparing copper–protoporphyrin–iron(III) complexes are presented. Comparison of the properties of these complexes with haematin and µ-oxo-dimeric haematin clearly shows that the complexes are distinct new species. Infrared spectroscopy provides evidence for the copper binding to protoporphyrin propionate groups. Mössbauer and e.s.r. spectroscopy provide evidence for high-spin iron(III) and copper(II). There is some evidence for interactions between the two metal nuclei. A possible structure is presented and its properties are discussed in terms of the known behaviour of cytochrome c oxidase.