Zn Protoporphyrin Research Articles

Zinc Protoporphyrin-Rich Pork Liver Homogenates as Coloring Ingredients in Nitrite-Free Liver Pâtés.

This study aimed to investigate the coloring ingredient potential of liver homogenates that form Zn protoporphyrin (ZnPP), a natural red pigment, after anaerobic incubation. Liver homogenates were used to develop nitrite-free sterile pork liver pâtés. These homogenates were applied in the formulation of pâtés directly or after centrifugation to obtain a pellet that was highly concentrated in ZnPP. Both the whole homogenate and its insoluble fraction were adjusted to pH 7.5 before their use in the formulation of pâtés with and without antioxidant (0.5% ascorbate plus 0.1% tocopherol) addition. Pâtés formulated with the whole homogenate showed color and texture characteristics that were similar to those of the positive control with nitrite. However, high levels of the insoluble fraction also led to pâtés with improved color characteristics but with a two-fold softened texture. Therefore, the form and amount of ZnPP added played roles in the final appearance of the product. The ZnPP pigment was more stable than heme in the sterilization treatment, and antioxidant addition proved to be unnecessary. The ZnPP-rich ingredients allowed for the preparation of nitrite-free cooked liver pâtés with a stable red color and could thus be potentially applied in other uncured cooked meat products.

UHPLC-MS/MS-based lipidomics for the evaluation of the relationship between lipid changes and Zn-protoporphyrin formation during Nuodeng ham processing

The effects of changes in lipids on the formation of Zn-protoporphyrin (ZnPP) during the processing of Nuodeng ham were analyzed using a lipidomics approach based on UHPLC-MS/MS. The Nuodeng ham samples had a strong fluorescence emission peak at 590 nm, and the fluorescence intensity increased with the processing time. Lipid profiles were mainly affected by processing time. A total of 5 lipid classes were detected, of which glycerophospholipids (GP) and glycerolipids (GL) were the most abundant lipids. Fifty differential lipid compounds were screened, which were mainly GP and GL. Correlation analysis showed that 13 differential lipid compounds were significantly positively correlated with ZnPP content, and they contained more linoleic acid and oleic acid branch chains. Meanwhile, all triglycerides (TG) were negatively correlated with heme content, and they were enriched in stearic acid and palmitic acid branch chains. These findings can deepen the understanding of the relationship between ZnPP and lipids.

Aptamer-Modified Homogeneous Catalysts, Heterogenous Nanoparticle Catalysts, and Photocatalysts: Functional "Nucleoapzymes", "Aptananozymes", and "Photoaptazymes".

Conjugation of aptamers to homogeneous catalysts ("nucleoapzymes"), heterogeneous nanoparticle catalysts ("aptananozymes"), and photocatalysts ("photoaptazymes") yields superior catalytic/photocatalytic hybrid nanostructures emulating functions of native enzymes and photosystems. The concentration of the substrate in proximity to the catalytic sites ("molarity effect") or spatial concentration of electron-acceptor units in spatial proximity to the photosensitizers, by aptamer-ligand complexes, leads to enhanced catalytic/photocatalytic efficacies of the hybrid nanostructures. This is exemplified by sets of "nucleoapzymes" composed of aptamers conjugated to the hemin/G-quadruplex DNAzymes or metal-ligand complexes as catalysts, catalyzing the oxidation of dopamine to aminochrome, oxygen-insertion into the Ar─H moiety of tyrosinamide and the subsequent oxidation of the catechol product into aminochrome, or the hydrolysis of esters or ATP. Also, aptananozymes consisting of aptamers conjugated to Cu2+ - or Ce4+ -ion-modified C-dots or polyadenine-stabilized Au nanoparticles acting as catalysts oxidizingdopamine or operating bioreactor biocatalytic cascades, are demonstrated. In addition, aptamers conjugated to the Ru(II)-tris-bipyridine photosensitizer or the Zn(II) protoporphyrin IX photosensitizer provide supramolecular photoaptazyme assemblies emulating native photosynthetic reaction centers. Effective photoinduced electron transfer followed by the catalyzed synthesis of NADPH or the evolution of H2 is demonstrated by the photosystems. Structure-function relationships dictate the catalytic and photocatalytic efficacies of the systems.

Nanoscale coordination polymers enabling antioxidants inhibition for enhanced chemodynamic therapy.

Reactive oxygen species (ROS) generation to induce cell death is an effective strategy for cancer therapy. In particular, chemodynamic therapy (CDT), using Fenton-type reactions to generate highly cytotoxic hydroxyl radical (•OH), is a promising treatment modality. However, the therapeutic efficacy of ROS-based cancer treatment is still limited by some critical challenges, such as overexpression of enzymatic and non-enzymatic antioxidants by tumor cells, as well as the low tumor targeting efficiency of therapeutic agents. To address those problems, biomimetic CuZn protoporphyrin IX nanoscale coordination polymers have been developed, which significantly amplify oxidative stress against tumors by simultaneously inhibiting enzymatic and non-enzymatic antioxidants and initiating the CDT. In this design, cancer cell membrane camouflaged nanoparticle exhibits an excellent homotypic targeting effect. After being endocytosed into tumor cells, the nanoparticles induce depletion of the main non-enzymatic antioxidant glutathione (GSH) by undergoing a redox reaction with GSH. Afterward, the redox reaction generated cuprous ion (Cu+) works as a CDT agent for •OH generation. Furthermore, the released Zn protoporphyrin IX strongly inhibits the activity of the typical enzymatic antioxidant heme oxygenase-1. This tetra-modal synergistic strategy endows the biomimetic nanoparticles with great capability for anticancer therapy, which has been demonstrated in both in vitro and in vivo studies.

Combined application of high-throughput sequencing and LC-MS/MS-based metabolomics to evaluate the formation of Zn-protoporphyrin in Nuodeng ham

In order to deepen the understanding of the formation mechanism of Zn-protoporphyrin (ZnPP), Nuodeng ham was used as the research object, and the effects of bacterial communities and small molecule metabolites on the formation of ZnPP during the processing of Nuodeng ham were analyzed by high-throughput sequencing and LC-MS/MS-based metabolomics. With the prolongation of processing time, the ZnPP content increased significantly, while the heme content decreased significantly. Compound curing agent significantly inhibited the formation of ZnPP and significantly decreased the heme content. The bacterial communities changed dynamically and the five bacterial genera were significantly positively correlated with ZnPP content. Forty-seven differential metabolites were obtained through screening, of which seventeen differential metabolites were significantly positively correlated with ZnPP content. Correlation analysis showed a strongly positive correlation between Staphylococcus, Delftia, Acinetobacter and these seventeen differential metabolites. These findings can provide a theoretical basis for further establishing the color control measures of Nuodeng ham.

Nanographene-based electrochemical sensors for ultrasensitive determination of sorbic acid from food.

Ultrasensitive determination of sorbic acid in food is essential for the assessment of the food quality. Therefore, two sensors based on nanographene decorated with gold nanoparticle paste modified with metal porphyrins (Zn protoporphyrin IX, and 2,3,7,8,12,13,17,18 octaethyl, 21H, 23H-porphirine Mn(III) chloride) were proposed for the determination of sorbic acid in food (bakery products and mayonnaise). Square-wave voltammetry was used for the characterization and validation of the proposed sensors. Response characteristics showed that the limits of detection for both sensors were 0.33µmol L-1 while the limits of quantification were 1.00µmol L-1. Both sensors can be used for the determination of sorbic acid in the concentration range 1-1000µmol L-1, the linear concentration range making them appropriate for the assay of sorbic acid in food. The highest sensitivity (0.35 nA/µmol L-1) was recorded when the sensor based on 2,3,7,8,12,13,17,18 octaethyl, 21H, 23H-porphirine Mn(III) chloride was used, proving the higher electrocatalytic effect of this electrocatalyst versus the one of the Zn protoporphyrin IX. High recoveries (values higher than 95.00%) and low RSD (%) values (lower than 5.00%) were recorded for both sensors when used for the determination of sorbic acid in bread and mayonnaise, proving the high reliability of the proposed sensors and method.

Heme oxygenase-1/carbon monoxide signaling participates in the accumulation of triterpenoids of Ganoderma lucidum.

Ganoderic triterpenoids (GTs) are the primary bioactive constituents of the Basidiomycotina fungus, Ganoderma lucidum. These compounds exhibit antitumor, anti-hyperlipidemic, and immune-modulatory pharmacological activities. This study focused on GT accumulation in mycelia of G. lucidum mediated by the heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling. Compared with the control, hemin (10 μmol/L) induced an increase of 60.1% in GT content and 57.1% in HO-1 activity. Moreover, carbon monoxide-releasing molecule-2 (CORM-2), CO donor, increased GT content by 56.0% and HO-1 activity by 18.1%. Zn protoporphyrin IX (ZnPPIX), a specific HO-1 inhibitor, significantly reduced GT content by 26.0% and HO-1 activity by 15.8%, while hemin supplementation reversed these effects. Transcriptome sequencing showed that HO-1/CO could function directly as a regulator involved in promoting GT accumulation by regulating gene expression in the mevalonate pathway, and modulating the reactive oxygen species (ROS) and Ca2+ pathways. The results of this study may help enhance large-scale GT production and support further exploration of GT metabolic networks and relevant signaling cross-talk.

Gated Dissipative Dynamic Artificial Photosynthetic Model Systems.

Gated dissipative artificial photosynthetic systems modeling dynamically modulated environmental effects on the photosynthetic apparatus are presented. Two photochemical systems composed of a supramolecular duplex scaffold, a photosensitizer-functionalized strand (photosensitizer is Zn(II)protoporphyrin IX, Zn(II)PPIX, or pyrene), an electron acceptor bipyridinium (V2+)-modified strand, and a nicking enzyme (Nt.BbvCI) act as functional assemblies driving transient photosynthetic-like processes. In the presence of a fuel strand, the transient electron transfer quenching of the photosensitizers, in each of the photochemical systems, is activated. In the presence of a sacrificial electron donor (mercaptoethanol) and continuous irradiation, the resulting electron transfer process in the Zn(II)PPIX/V2+ photochemical module leads to the transient accumulation and depletion of the bipyridinium radical-cation (V·+) product, and in the presence of ferredoxin-NADP+ reductase and NADP+, to the kinetically modulated photosynthesis of NADPH. By subjecting the mixture of two photochemical modules to one of two inhibitors, the gated transient photoinduced electron transfer in the two modules is demonstrated. Such gated dissipative process highlights its potential as an important pathway to protect artificial photosynthetic module against overdose of irradiance and to minimize photodamage.

Supramolecular Metalloporphyrin Assemblies Mediated By a Hexameric Hemoprotein Unit

Supramolecular protein assemblies provide unique structures, thermodynamic behaviors, and attractive functions derived from reversible interactions between protein-based building blocks. In nature, there are many kinds of unique protein assemblies including rings, sheets, tubes and cages, which are formed by multiple interactions such as electrostatic interactions, hydrophobic contacts, coordination etc. These beautiful composites have encouraged chemists to generate new bionanomaterials and then a lot of artificial protein assemblies have been reported over last decade.Hemoproteins possess a cofactor, protoporphyrin IX iron complex (heme b), which is bound in the heme pocket via Fe–axial ligand coordination and non-covalent interaction with large affinities. It is well known that the heme cofactor is removable from the heme pocket under acidic conditions to yield a corresponding apoprotein. Furthermore, the apoprotein can be converted to a reconstituted protein upon the addition of an artificial cofactor [1]. Our group has focused on a specific interaction between heme and the heme pocket of a hemoprotein to construct supramolecular hemoprotein assembles.A hexameric tyrosine-coordinated heme protein (HTHP) provides a ring structure containing six domains. Each domain has a heme-binding site named a heme popcket. The heme cofactors are bound in the heme pockets, showing a circular aggregate of the heme cofactor within protein matrix. First, we replaced the heme cofactor with Zn protoporphyrin IX (ZnPP) or Zn chlorin e6 (ZnCe6) to obtain a photosensitizer assembly mediated by protein. The measurement of femtosecond transient absorption spectra upon pulse irradiation showed a rapid singlet-singlet annihilation within a few picoseconds for the reconstituted HTHP. This finding supports the fact that the photoinduced energy migration occurs within the protein. Furthermore, the fluorescence quenching efficiency determined by the addition of methyl viologen as an electron acceptor also support the existence of the photoinduced energy migration for the photosensitizer-inserted protein. These findings are expected to lead to development of new protein-based artificial light harvesting systems [2].Furthermore, our group recently tried to construct supramolecular assembly of HTHP to obtain 1D, 2D and 3D protein assembled structures formed by non-covalent multi interaction between modified HTHP. In the case of the linear HTHP assembly, we introduced pyrene moiety onto the surface of HTHP and then provided the stable dimer of HTHP [3]. The 2D and 3D structures have already been prepared as an artificial light-harvesting system [4].In this presentation, we want to report photochemical property of HTHP reconstituted with the Zn photosensitizer and the trial of the construction of the highly dimensional HTHP assembly.

Fluorescent Analogue of Hexa-coordinate Globins to Monitor Accessibility of the Heme Pocket

Neuroglobin (Ngb), cytoglobin (Cygb) and globin X (GbX) belong to the family of vertebrate hexa-coordinate globins. Recent studies have proposed that these globins carry out various function including ROS/RNS scavenger and in vivo studies have indicated the role of Cygb and Ngb in carcinogenesis, cell proliferation, apoptosis and cell survival under hypoxic conditions. Globin X was found to be important for the reduction of nitrite to nitric oxide in fish red blood cells. It was shown that the internal disulfide bond that is unique to hexa-coordinate vertebrate globins modules the heme iron affinity for oxygen in Ngb and Cygb. To probe the mechanism of the ligand migration in Cygb, Ngb and GbX, we replaced the non-fluorescent Fe protoporphyrin IX (FePPIX) with the fluorescent analogue Zn protoporphyrin IX (ZnPPIX) and characterized the ligand migration by monitoring the quenching of the ZnPPIX fluorescent emission by methyl viologen and the quenching of the ZnPPIX triplet state by oxygen. We observed an increase in the Ksv value for methyl viologen quenching in protein variants that are missing the internal disulfide bond, suggesting that the presence of the intraprotein disulfide bond regulates the accessibility of the heme binding pocket. Interestingly the lifetime of the triplet state for ZnPPIX incorporated in hexacoordinate vertebrate globins is not sensitive to changes in the heme environment as the observed lifetime is comparable to that determined previously for myoglobin.

Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System.

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

Is the Iron Treatment Important for Lowering Erythrocyte Zinc in Non-Dialyzed Patients with Chronic Kidney Disease?

Introduction: Zinc (Zn) protoporphyrin (ZPP) presents in red blood cells (RBC) in small amounts, but increases in iron deficiency or lead poisoning. Formation of heme - a component of hemoglobin - finished with insertion of an iron into the molecule of protoporphyrin. In iron deficiency, or inhibition of iron insertion (lead poisoning), zinc replaces iron to form ZPP, which cannot bind to oxygen. Plasma zinc is lower in patients with chronic kidney diseases (CKD), but it’s not yet clear whether it’s a real deficit or is due to redistribution. Aim: The aim of this study was to evaluate the effects of iron supplementation on zinc redistribution in the body tissues of non-dialyzed chronic kidney disease (CKD) patients. Material and Methods: Zn and Fe status of 38 non-dialyzed patients with iron-deficit anemia (17 males; 21 females);mean GFR - 43.3 mL/min(32.5 to 57.3 mL/min), was evaluated before and after 5 i.v. injections of 100 mg iron each. The following parameters were tested: RBC and plasma Zn, ZPP, ferritin (F), transferrin saturation (TFS), and total iron (Fe). Results: F and TFS increased from 86.5+/-61.2 ng/mL to 132.4+/-64.3 ng/mL and from 21.5+/-9.4% to 26.8+/-8.3% (P<0.05), respectively, after iron supplementation. Absolute iron deficiency (ferritin <100 g/L and TFS < 20%) was present in 44% of the patients and decreased to 25% after iron treatment. After supplementation, patients with low plasma zinc decreased from 49.5% to 30.8% (p N.S.). At start of the study 29 of the patients had high erythrocyte zinc. After iron treatment, erythrocyte zinc decreased significantly in 24 patients (p< 0.001), and only a weak lowering was observed in ZPP (p N.S.). Conclusions: The study suggests that iron deficiency may participate in the inadequate distribution of zinc in patients with CKD and iron supplementation may decrease the abnormal elevated erythrocyte zinc levels.

Electron spin polarization transfer induced by triplet-radical interactions in the weakly coupled regime.

We report the observation of electron spin polarization transfer from the triplet state of a porphyrin to a weakly coupled nitroxide radical in a mutant of human neuroglobin (NGB). The native iron-containing heme substrate of NGB has been substituted with Zn(ii) protoporphyrin IX and the nitroxide has been attached via site-directed spin labeling to the Cys120 residue. A reference synthetic polypeptide with free base tetraphenylporphyrin and a nitroxide bound to it is also studied. In both systems the nitroxide and the porphyrin are held at a fixed distance of approximately 2.4 nm. The transient EPR data of the NGB sample show that the triplet state of Zn(ii) protoporphyrin acquires significant net polarization, which is attributed to the dynamic Jahn-Teller effect. As the spin polarization of the protoporphyrin triplet state decays, a polarized EPR signal of the nitroxide arises. In contrast, the free base porphyrin in the reference polypeptide does not acquire net polarization and no polarization of the nitroxide label is observed. This is likely a result of the fact that the porphyrin is not Jahn-Teller active because of its lower symmetry. A perturbation theory treatment suggests that in the NGB sample, the polarization of the radical occurs by the transfer of net polarization from the triplet state. This process is also enhanced by the spectral broadening caused by the back and forth transitions associated with the dynamic Jahn-Teller effect. We propose that the novel transfer of polarization to the radical could be exploited to enhance the sensitivity of light-induced dipolar spectroscopy experiments.

Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein.

Light‐induced pulsed EPR dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites. Here we employ orthogonal spin labels, a chromophore triplet state and a stable radical, to carry out distance measurements in singly nitroxide‐labeled human neuroglobin. We demonstrate that Zn‐substitution of neuroglobin, to populate the Zn(II) protoporphyrin IX triplet state, makes it possible to perform light‐induced pulsed dipolar experiments on hemeproteins, extending the use of light‐induced dipolar spectroscopy to this large class of metalloproteins. The versatility of the method is ensured by the employment of different techniques: relaxation‐induced dipolar modulation enhancement (RIDME) is applied for the first time to the photoexcited triplet state. In addition, an alternative pulse scheme for laser‐induced magnetic dipole (LaserIMD) spectroscopy, based on the refocused‐echo detection sequence, is proposed for accurate zero‐time determination and reliable distance analysis.

Inhibition of TREM1 reduces inflammation and oxidative stress after spinal cord injury (SCI) associated with HO-1 expressions

Spinal cord injury (SCI) is a devastating event, leading to the progression of chronic neuropathic pain syndrome. Triggering receptor expressed on myeloid cells 1 (TREM1) is an innate immune receptor expressed on neutrophils and monocytes/macrophages. TREM1 enhances inflammatory response in various models of diseases, but its significance in SCI remains unclear. In the present study, we attempted to explore the effects of TREM1 on the regulation of SCI. Spinal cord contusion injury was performed in wild type (WT) and TREM1-knockout (TREM1KO) mice, and real time-quantitative PCR (RT-qPCR), western blot, and immunofluorescent (IF) staining were used to calculate TREM1, inflammation and oxidative stress in spinal cord tissues 42 days after SPII. In vitro, astrocytes (AST) and BV2 cells were transfected TREM siRNA or the negative control (NC) siRNA to knockdown (KD) TREM1 expressions, followed by lipopolysaccharide (LPS) stimulation to verify the role od TREM1 in modulating SPI. The results suggested that TREM1 was highly expressed in the spinal cord tissues of WT mice after SCI. TREMKO mice exhibited improved locomotor function, mechanical and thermal hypersensitivity in the hindpaws after SCI. In addition, peripheral nerve injury-related biomarkers were down-regulated by TREM1KO in SCI mice. TREM1KO increased NeuN-stained cells, and decreased GFAP and Iba-1 expressions in spinal cord tissues of mice after SCI. TREM1KO mice showed reduced expressions of inflammation-related regulators in the injured spinal cord. Further, toll like receptors (TLR-2, -3, -4, and -9), p-IκBα and p-nuclear factor-kappa B (NF-κB) protein expression levels were markedly decreased by TREM1KO in mice after SCI injury. Moreover, TREM-deficiency suppressed oxidative stress markers, while enhanced anti-oxidants, such as superoxide dismutase-1 (SOD1), NAD(P)H:quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1) and nuclear factor E2–related factor 2 (Nrf2) in the injured spinal cord. The in vivo results mentioned above were verified in LPS-stimulated AST and BV2 cells in vitro. Of note, the in vitro results also demonstrated that suppressing HO-1 expressions using Zn-protoporphyrin (ZnPP) abrogated TREM1KD-reduced inflammation, oxidative stress and glial cells activation. The results above demonstrated that suppressing TREM1 expressions markedly improved the outcome of SCI, most likely through reducing inflammation and oxidative stress at least partly regulated by HO-1 expressions. TREM1 inhibition might be therefore has potential as a therapeutic target after SCI.

Preparation and Photochemical Properties of Supramolecular Hemoprotein Assemblies

A series of familiar hemoproteins such as myoglobin, horseradish peroxidase, cytochrome P450 possess a cofactor, protoporphyrin IX iron complex (heme b), which is bound in the heme pocket via Fe–axial ligand coordination and non-covalent interaction with large affinities. The cofactor is removable from the heme pocket under acidic conditions to yield a corresponding apoprotein. Furthermore, the protein reconstitution is enabled by incorporating an artificial cofactor into the apoprotein. Over the last decade, we have focused on the heme-substitution with several porphyrinoid metal complexes.1) In addition, we have introduced a heme moiety onto the hemoprotein surface via a covalent linkage to construct a supramolecular protein assembly via interprotein heme–heme pocket interaction. Here, we present a topic of hemoprotein assembly formed by the reconstitutional method. A hexameric tyrosine-coordinated heme protein (HTHP) containing photosensitizer molecules was prepared to construct a biomolecule having engineered photochemical properties.2) Zn protoporphyrin IX (ZnPP) or Zn chlorin e6 (ZnCe6) was inserted into the apoprotein of HTHP to yield the reconstituted protein, which maintained the intrinsic hexameric structure of HTHP. Femtosecond transient absorption measurements by a flash photolysis system suggest the occurrence of rapid singlet–singlet annihilation within a few picoseconds for each protein completely reconstituted with the photosensitizer molecules. This finding supports the fact that the photo-induced energy migration occurs within the protein with the zinc complex. The fluorescence quenching efficiency determined by the addition of methyl viologen as an electron acceptor for the completely reconstituted protein with ZnPP is 2.3 fold-higher than that of the partially photosensitizer-inserted protein where only one molecular of ZnPP is inserted into the hexamer. This indicates that the energy migration occurs among the photosensitizers bound within the protein matrix. These findings are expected to lead to development of new protein-based artificial light harvesting systems. Furthermore, our group recently tried to introduce five fluorescein molecules and one Texas Red on the surface of the cysteine-containing HTHP mutant with ZnPP. Upon the irradiation of fluorescein at 593 nm, the energy transfer from fluorescein was observed by monitoring the emission of Texas Red at 615 nm.3) We found two pathways of the energy transfer; one is the direct energy transfer from fluorescein to Texas Red and another is the energy transfer via ZnPP. The ratio of the pathways is 39 : 61, indicating that the energy is collected at the Texas Red molecule as a funnel-like bottom for light-harvesting. This system will serve as a new model to demonstrate a light-harvesting event. REFERENCES 1) Hayashi, T. In Handbook of Porphyrin Science; Kadish, K. M.; Smith, K. M.; Guilard, R., Eds.; World Scientific Publishing: Singapore, 2010; Vol. 5, pp 1–69. 2) Oohora, K.; Mashima, T.; Ohkubo, K.; Fukuzumi, S.; Hayashi, T. Chem. Commun. 2015, 51, 11138. 3) Mashima, T.; Oohora, K.; Hayashi, T. Phys. Chem. Chem. Phys. 2017 in press.

Probing the Highly Efficient Electron Transfer Dynamics between Zinc Protoporphyrin IX and Sodium Titanate Nanosheets.

Sodium titanate nanosheets (NaTiO2 NS) have been prepared by a new method and completely characterized by TEM, SEM, XRD, EDX, and XPS techniques. The sensitization of nanosheets is carried out with Zn protoporphyrin IX (ZnPPIX). The emission intensity of ZnPPIX is quenched by NaTiO2 NS, and the dominant process for this quenching has been attributed to the process of photoinduced electron injection from excited ZnPPIX to the nanosheets. Time resolved fluorescence measurement was used to elucidate the process of electron injection from the singlet state of ZnPPIX to the conduction band of NaTiO2 NS. Electron injection from the dye to the semiconductor is very fast (ket ≈ 10(11) s(-1)), much faster than previously reported rates. The large two-dimensional surface offered by the NaTiO2 NS for interaction with the dye and the favorable driving force for electron injection from ZnPPIX to NaTiO2 NS (ΔGinj = -0.66 V) are the two important factors responsible for such efficient electron injection. Thus, NaTiO2 NS can serve as an effective alternative to the use of TiO2 nanoparticles in dye sensitized solar cells (DSSCs).

Effects of Processing Temperature on Color Properties of Dry-Cured Hams Made without Nitrite.

Dry cured hams were investigated for their ability to develop red color even at low temperature (3–4 °C) and in the absence of added nitrites; results were compared with those obtained from nitrite-free hams made at conventional warm maturing temperatures. Colorimetric parameters (L*, a*, b*, and hue) and concentration of the main pigments Zn protoporphyrin IX (ZnPP) and heme were measured at three stages of preparation (six, nine, and 12 months), showing that red color was successfully formed at low temperatures, though at a slower rate and less intensively than under warm conditions. Major differences in the pattern of color development were found with the two processing temperatures. While the typical features of an enzyme-dependent mechanism, with a progressive drop in enzyme activity paralleling the synthesis of Zn protoporphyrin IX, were observed at warm temperatures, the same did not occur in cold-made hams, where the enzyme activity was almost unchanged throughout the process. These results, along with data from a descriptive sensory analysis, are supportive of a non-enzymatic mechanism leading to ZnPP (hence the red color) under cold conditions, with an estimated three-month delay compared with nitrite-free hams manufactured in a warm maturing regimen.

(Invited) Construction of Hemoprotein Assembly Via Heme-Heme Pocket Interaction and Its Photochemical Property

Hemoproteins are metalloproteins which possess iron porphyrin as a prosthetic group (cofactor). These proteins have received much attention not only as biocatalysts but also as promising building blocks for development of new types of biomaterials. Particularly, the heme cofactor in several hemoproteins such as myoglobin, horseradish peroxidase, cytochrome P450, and cytochrome b 562 are replaceable with an artificial heme analog. To functionalize the hemoproteins, our group has focused on the cofactor-substitution with several porphyrinoid metal complexes.1) Furthermore, we have recently introduced a heme moiety onto the myoglobin surface via covalent linkage to obtain a supramolecular protein polymer via interprotein heme–heme pocket interaction.2) In this presentation, we report the recent results of the hemoprotein assembly which is a model of a light-harvesting system.3) A hexameric tyrosine-coordinated heme protein (HTHP) containing photosensitizer molecules was prepared in an effort to construct a biomolecule with engineered photochemical properties. Zn protoporphyrin IX (ZnPP) and Zn chlorin e6 (ZnCe6) are inserted into the apoprotein of HTHP to yield reconstituted proteins which maintain the original hexameric structure of HTHP. Femtosecond transient absorption measurements indicate the occurrence of rapid singlet–singlet annihilation within a few picoseconds for each protein completely reconstituted with the six photosensitizer molecules. This finding supports the fact that the photo-induced energy migration occurs whtin the protein with the zinc complex. The fluorescence quenching efficiencies provided by methyl viologen as an electron acceptor for the completely reconstituted proteins with ZnPP and ZnCe6 are 2.3 and 2.6 fold-higher than that of the corresponding partially photosensitizer-inserted proteins, respectively. This indicates that energy migration occurs among the photosensitizers bound within the protein matrices. These findings are expected to lead to development of new protein-based artificial light harvesting systems. 1) Hayashi, T. In Coordination Chemistry in Protein Cages; Ueno, T.; Watanabe, Y., Eds.; Wiley: Hoboken, US, 2013; Chapter 4, pp 87–110. 2) Oohora, K.; Onoda, A.; Hayashi, T. Chem. Commun. 2012, 48, 11714–11726. 3) Oohora, K.; Mashima, T.; Ohkubo, K.; Fukuzumi, S.; Hayashi, T. Chem. Commun. 2015, 51, 11138–11140.

Haem oxygenase 1 expression is associated with prognosis in cholangiocarcinoma patients and with drug sensitivity in xenografted mice.

Haem oxygenase-1 (HO-1) plays important roles in cytoprotection and tumour growth. Cholangiocarcinoma (CCA) is a deadly malignancy with very poor prognosis. The role of HO-1 in tumour progression in CCA up to now has been relatively unexplored, thus, its possible therapeutic implications in CCA have been investigated here. HO-1 expression in tumour tissues from 50 CCA patients was determined by immunohistochemical analysis and its association with survival time was evaluated using the Kaplan-Meier method. Its role in CCA cells in vitro was evaluated by transwell and wound healing assays and suppression of HO-1 expression by siRNA. Effects of HO-1 inhibition on gemicitabine (GEM)-mediated tumour suppression was evaluated in nude mice xenografted with CCA cells. HO-1 expression was inversely associated with median overall survival time. Hazard ratio of patients with high HO-1 expression was 2.42 (95% CI: 1.16-5.08) with reference to low expression and HO-1 knock-down expression inhibited transwell cell migration. Suppression of HO-1 by Zn-protoporphyrin (ZnPP) enhanced cytotoxicity to GEM in CCA cells, validated in CCA xenografts. Treatment with GEM and ZnPP almost completely arrested tumour growth, whereas treatment with only a single reagent, retarded it. Tumour inhibition was associated with reduction in expression of Ki-67 and microvascular density, and enhanced p53 and p21 immunohistochemical staining. High HO-1 expression was associated with poor prognosis of CCA. Synergistic role of HO-1 inhibition in chemotherapy of CCA is a promising insight for treatment of this tumour and warrants further investigation.