Structural Studies on Porphyrin–PNA Conjugates in Parallel PNA:PNA Duplexes: Effect of Stacking Interactions on Helicity

Parallel PNA:PNA duplexes were synthesized and conjugated with meso‐tris(pyridyl)phenylporphyrin carboxylic acid at the N‐terminus. The introduction of one porphyrin unit was shown to affect slightly the stability of the PNA:PNA parallel duplex, whereas the presence of two porphyrin units at the same end resulted in a dramatic increase of the melting temperature, accompanied by hysteresis between melting and cooling curves. The circular dichroism (CD) profile of the Soret band and fluorescence quenching strongly support the occurrence of a face‐to‐face interaction between the two porphyrin units. Introduction of a L‐lysine residue at the C‐terminal of one strand of the parallel duplex induced a left‐handed helical structure in the PNA:PNA duplex if the latter contains only one or no porphyrin moiety. The left‐handed helicity was revealed by nucleobase CD profile at 240–280 nm and by the induced‐CD observed in the presence of the DiSC₂(5) cyanine dye at ~500–550 nm. Surprisingly, the presence of two porphyrin units led to the disappearance of the nucleobase CD signal and the absence of CD exciton coupling within the Soret band region. In addition, a dramatic decrease of induced CD of DiSC₂(5) was observed. These results are in agreement with a model where the porphyrin–porphyrin interactions cause partial loss of chirality of the PNA:PNA parallel duplex, forcing it to adopt a ladder‐like conformation. Chirality 27:864–874, 2015. © 2015 Wiley Periodicals, Inc.     

Determination of trace α‐fetoprotein variant by affinity adsorption solid substrate‐room temperature phosphorimetry

The 3.5‐generation dendrimers (3.5G‐D)–porphyrin (P) dual luminescent molecule (3.5G‐D–P) was used to label concanavalin agglutinin (Con A); the product of the reaction is 3.5G‐D–P–Con A. A new method for the determination of trace AFP‐V by affinity adsorption solid substrate–room temperature phosphorimetry (AA‐SS–RTP) was established, based on the room temperature phosphorescence (RTP) property of the product on polyamide membrane (PAM) substrate and the specific affinity adsorption (AA) reaction between 3.5G‐D–P–Con A and α‐fetoprotein variant (AFP‐V), which caused the RTP of the system to be sharply enhanced, the ΔIp was linearly correlated to the content of AFP‐V. The sensitivity of the method was obviously high. It could accurately detect the content of AFP‐V in serum. The results were tallied well with those obtained by the ELISA method. Copyright © 2008 John Wiley & Sons, Ltd.     

Ironing out the distribution of [2Fe-2S] motifs in ferrochelatases

Heme, a near ubiquitous cofactor, is synthesized by most organisms. The essential step of insertion of iron into the porphyrin macrocycle is mediated by the enzyme ferrochelatase. Several ferrochelatases have been characterized, and it has been experimentally shown that a fraction of them contain [2Fe-2S] clusters. It has been suggested that all metazoan ferrochelatases have such clusters, but among bacteria, these clusters have been most commonly identified in Actinobacteria and a few other bacteria. Despite this, the function of the [2Fe-2S] cluster remains undefined. With the large number of sequenced genomes currently available, we comprehensively assessed the distribution of putative [2Fe-2S] clusters throughout the ferrochelatase protein family. We discovered that while rare within the bacterial ferrochelatase family, this cluster is prevalent in a subset of phyla. Of note is that genomic data show that the cluster is not common in Actinobacteria, as is currently thought based on the small number of actinobacterial ferrochelatases experimentally examined. With available physiological data for each genome included, we identified a correlation between the presence of the microbial cluster and aerobic metabolism. Additionally, our analysis suggests that Firmicute ferrochelatases are the most ancient and evolutionarily preceded the Alphaproteobacterial precursor to eukaryotic mitochondria. These findings shed light on distribution and evolution of the [2Fe-2S] cluster in ferrochelatases and will aid in determining the function of the cluster in heme synthesis.     

A novel chemiluminescent flow‐injection analysis of transferrin by its reduction of the luminol—hydrogen peroxide reaction catalysed by meso‐tetra‐(3‐methoxyl‐4‐hydroxyl) phenyl manganese porphyrin

Based on the inhibition effect of transferrin (Tf) on the reaction of the luminol–hydrogen peroxide (H₂O₂) chemiluminescence (CL) system, catalysed by meso‐tetra‐(3‐methoxyl‐4‐hydroxyl) phenyl manganese porphyrin (MnP) as a mimetic enzyme of peroxides, a sensitive flow‐injection CL method has been developed for the determination of Tf in an alkaline medium. The CL reaction was carefully investigated by examining the variations of reaction conditions. Under optimum conditions, the linear range for the determination of transferrin was 0.04–20.0 μg/mL and the detection limit was 1.62 ng/mL. This proposed method was sensitive, convenient and simple, and has been successfully applied to the determination of transferrin in a serum sample. Copyright © 2011 John Wiley & Sons, Ltd.     

Chemiluminescent measurement of oxalate in serum by detection of hydrogen peroxide generated through oxalate oxidase

Today, chemiluminescence detection reactions have become popular in analytical biochemistry essentially due to their high sensitivity. A chemiluminescent synthetic system (luminol/porphyrin) was successfully used to measure serum oxalate by determination of hydrogen peroxide generated through oxalate oxidase (EC 1.2.3.4.). This new method is efficient and simple, highly sensitive and the results obtained in normal adult subjects are in good agreement with those of approved methods. This original application of such a chemiluminescent system allowed us to achieve a sensitive serum oxalate assay (detection limit of 0.2 μmol/L) characterized by a low serum volume (200 μL) required for analysis. © 1997 John Wiley & Sons, Ltd.     

Catalytic oxidation of alkenes by manganese(III) porphyrin-encapsulated Al, V, Si-mesoporous molecular sieves

Cationic manganese-porphyrin, [meso-tetrakis(4-trimethylammoniophenyl)porphyrinato]manganese(III) pentachloride (MnTAPP), has been prepared and encapsulated into mesoporous molecular sieves Al-MCM-41 and V-MCM-41, containing different amounts of Al and V, respectively. The catalytic activities of these heterogeneous materials were tested in the liquid phase oxidation of cyclohexene and styrene in acetonitrile with iodosylbenzene (PhIO) as oxygen source. Both types of catalysts were active in the oxidation reaction. MnTAPP encapsulated in Al-MCM-41 produces allylic oxidation products alone and no epoxide with styrene was found. However, it produces both epoxide and allylic oxidation products with cyclohexene. At the same time, MnTAPP encapsulated in V-MCM-41 produces epoxide as major product and little allylic oxidation product with styrene, while both epoxide and allylic oxidation products were obtained with cyclohexene. It is suggested that the regioselective effect is due to relatively more acidic Al-MCM-41 than V-MCM-41 which could make the CC bond unreactive towards epoxidation and produces allylic oxidation product. With increasing Al or V content in the support, the porphyrin loading was found to increase, which in turn increases the catalytic activity of the heterogeneous systems. The heterogeneous catalysts were reused for three times. The selectivity of these heterogeneous catalysts does not change appreciably even after three times of reusing, but their catalytic activity decreases marginally. This may be attributed to catalyst leaching and/or decomposition of MnTAPP complex under the reaction conditions.     

Porphyrin Binding to Gun4 Protein,Facilitated by a Flexible Loop,Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.     

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat

The activities of four heme-biosynthetic enzymes, -aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).Abbreviations ALA o-aminolevulinic acid - DAB 4-dimethyl aminoazobenzene - HpD hematoporphyrin derivative - 4NQO 4-nitroquinoline 1-oxide - PBG porphobilinogen