Role of immersion refractometry for investigating laser-induced effects in cells.

The broad background of scattered light observed in spectra of cell suspensions is reduced by factors of up to 20 by immersion refractometry allowing for improved spectroscopic determination of the absorption properties of cells in the 325-820 nm range. Refractive-index matched spectra of E. coli C1a exhibit a set of resonant features near 422, 561, and 582 nm. Exposure wavelengths are chosen based on this spectrum and cell viability is investigated in E. coli suspensions exposed to 350, 400, 422, 440, and 700 nm radiation delivered in nanosecond pulses with total doses from 500 millijoules to 60 Joules. We observe a loss in cell viability for doses greater than 1 Joule at 422 nm and for all doses at other wavelengths; exposures of less than 1 Joule at 422 nm enhance growth. Excluding exposures at wavelengths within the resonant feature, longer wavelengths are less effective at reducing the viability of E. coli C1a. This indicates the occurrence of at least two absorption processes.     

2,7,12,18-四甲基-13,17-二(3-羟基丙基)卟啉的合成及其光敏化活性

由氯化高铁次卟啉经脱铁、酯化、还原合成了光敏剂２，７，１２，１８－四甲基－１３，１７－二（３－羟基丙基）卟啉，并测定其光敏活性     

卟啉生物化学研究概况

本文在卟啉的化学结构和分类、化学检测方法、化学合成以及卟啉生物合成等方面对卟啉生物化学的研究概况进行综述。     

四磺酸卟啉镁对小鼠Lewis肺癌的放射增敏效应

探讨四磺酸卟啉镁的放射增敏效应。材料与方法将荷Ｌｅｗｉｓ肺癌的Ｃ５７ＢＬ／６小鼠随机分为４组：对照组、单药组、单放组及用药加放射组。其中单药组和用药加放射组小鼠腹腔单次注射四磺酸卟啉镁１３．７５ｍｇ／ｋｇ体重，单放组和用药加放射组各分１０、１５、２０Ｇｙ３个剂量小组进行单次放射，每组各８只鼠，结果（１）当肿瘤体积达原照射体积４倍时，测得四磺酸卟啉镁的增敏比为１．４５～１．７０，平均１．５８。（２）各组小鼠生存时间无显著差异。结论四磺酸卟啉镁对Ｌｅｗｉｓ肺癌具有放射增敏效应。     

卟啉及其类似物在HPLC固定相中的应用

介绍了卟啉及其类似物衍生固定相的特征和制法,综述了这类固定相在HPLC中的应用和分离机制.     

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

The identity of a metal center often defines enzymatic activity, and swapping the native metal for an alternative one or introducing a new metal center has profound effects. More generally, the chemical utility of natural cofactors has inspired decades of study into synthetic analogs with distinct properties, and researchers have subsequently sought straightforward ways to put these novel cofactors back into proteins (1). Substituted metalloenzymes constitute one of the simplest cases. Changing the identity of the metal ion in metalloproteins has enabled powerful spectroscopic and functional studies of these proteins (2–10) in addition to new biocatalytic activities (11–20). However, most methods for producing such proteins with new-to-nature cofactors are limited by the inability to produce the novel protein–cofactor complex in vivo.Hemoproteins, in particular, have been studied through metal substitution because of their important biological functions and utility as biocatalysts. Heme is a ubiquitous and versatile cofactor in biology, and heme-dependent proteins serve essential gas sensing functions (21), metabolize an array of xenobiotic molecules (22), and perform synthetically useful oxygen activation and radical-based chemistry (23). Metal-substituted hemoproteins have enabled key spectroscopic studies of hemoprotein function and the development of biocatalysts with novel reactivity. For example, electron paramagnetic resonance (EPR) studies on cobalt-substituted sperm whale myoglobin (CoMb) enabled detailed characterization of the paramagnetic CoMbO₂ complex (3, 4, 24, 25). In analogous oxygen-binding studies in CoMb and cobalt-substituted hemoglobin (5, 6, 26), resonance Raman was used to identify the O–O stretching mode because cobalt-substituted proteins exhibit enhancement of this vibrational mode compared to the native iron proteins.Metal substitution has a profound effect on catalytic activity of hemoproteins, enabling numerous synthetic applications. Substitution of the native iron for cobalt in several hemoproteins, including a thermostable cytochrome c variant, enabled the reduction of water to H₂ under aerobic, aqueous conditions (27–29). Reconstitution of apoprotein with selected metalloporphyrins has been used to generate metal-substituted myoglobin and cytochrome P450s variants. These enzymes were effective as biocatalysts for C–H activation and carbene insertion reactions (11–14). In a tour de force of directed evolution, which required purification and cofactor reconstitution of each individual variant, Hartwig and coworkers generated a cytochrome P450 variant that utilizes a nonnative Ir(Me)mesoporphyrin cofactor to perform desirable C–H activation chemistry (14). These activities may not be unique to the Ir-substituted protein, as synthetic cobalt porphyrin complexes have been shown to mediate a variety of Co(III)-aminyl and -alkyl radical transformations, including C–H activation (30–32). Indeed, a number of cobalt porphyrin carbene complexes display significant carbon-centered radical character (33–35), whereas the corresponding Fe-porphyrin complexes are closed shell species (36, 37), indicating that cobalt porphyrins may possess distinct, complementary modes of reactivity (38–40).Inspired by these applications, researchers have sought strategies for generating metal-substituted hemoproteins. For many metalloproteins, metal substitution is carried out by removal of the native metal with a chelator and replacement with an alternate metal of similar coordination preference. This method is inapplicable to hemoproteins, as porphyrins do not readily exchange metal ions. Consequently, diverse methods have been employed to make metal-substituted hemoproteins (41–46). Early on, copper, cobalt, nickel, and manganese-substituted horseradish peroxidase (HRP) were prepared by a multistep process that subjected protein to strong acid and organic solvents (41, 42). Variations of this method have been used repeatedly (24, 43, 47–49). However, this method is applicable only to a narrow range of hemoproteins that tolerate the harsh treatment. With the advent of overexpression methods, significant improvement of metalloporphyrin-substituted protein yield was achieved by direct expression of the apoprotein and reconstitution with the desired metalloporphyrin in lysate prior to purification (50). Although this approach has many virtues, direct expression of apoprotein is ineffective for many hemoproteins, again limiting the utility of this method.As an alternative to the above in vitro approaches, researchers have pursued systems for direct in vivo expression of metal substituted hemoproteins. Two specialty strains of Escherichia coli (E. coli) were engineered to incorporate metalloporphyrin analogs from the growth medium into hemoproteins during protein expression. The engineered RP523 strain cannot biosynthesize heme and bears an uncharacterized heme permeability phenotype. Together, these two features enable this strain to assimilate and incorporate various metalloporphyrins into overexpressed hemoproteins with no background heme incorporation (44, 51–53). However, heme auxotrophy makes RP523 cells exceedingly sensitive to O₂, and, in many situations, RP523 cultures must be grown anaerobically. An alternative BL21(DE3)-based engineered strain harbors a plasmid bearing the heme transporter ChuA, which facilitates import of exogenous heme analogs (45). Production of metalloporphyrin-substituted protein with this ChuA-containing strain relies on growth in iron-limited minimal media, thereby diminishing heme biosynthesis. This method was used successfully to express metal-substituted versions of the heme domain of cytochrome P450 BM3 (45) and several myoglobin variants (11, 12). Because these cells biosynthesize a small quantity of their own heme, they are far more robust than the RP523 cells. Unfortunately, this advantage comes at the cost of increased heme contamination in the product protein (2 to 5%) (45).A set of intriguing papers reported the production of cobalt-substituted human cystathionine β-synthase (CoCBS) that relies on the de novo biosynthesis of CoPPIX from CoCl₂ and δ-aminolevulinic acid (δALA), a biosynthetic precursor to heme (46, 54). This method yielded significant amounts of CoCBS—albeit with modest heme contamination (7.4%)—sufficient for spectroscopic and functional characterization of the CoPPIX-substituted protein (8, 46). As cobalt is known to be toxic to E. coli, the researchers passaged the CBS expression strain through cobalt-containing minimal media for 12 d, enabling the cells to adapt to high concentrations of cobalt prior to protein expression. It is plausible that this serial passaging alters the E. coli cells, enabling the biosynthesis of CoPPIX and in vivo production of metal-substituted protein. The adaptation process is slow (>10 d), and it is unknown how genomic instability under these mutagenic conditions affects the reproducibility of this passaging approach.The possibility of facile CoPPIX production is particularly attractive for future biocatalysis efforts. As described above, synthetic cobalt porphyrins have been shown to perform a range of radical-mediated reactions. The ability to produce a CoPPIX center in vivo may enable engineering these unusual reactivities via directed evolution in addition to spectroscopic applications. We therefore set out to explore the unusual phenotype of CoPPIX production by E. coli and to ascertain whether it was possible to efficiently biosynthesize cobalt-containing hemoproteins in vivo from a single “generalist” cell line. Our goal was to achieve an efficient and facile method of cobalt-substituted hemoprotein production with minimal contamination of the native cofactor. Herein, we report the surprising discovery that native E. coli BL21(DE3) can biosynthesize a new-to-nature CoPPIX cofactor (Fig. 1). We use this insight to produce cobalt-substituted hemoproteins in vivo without requirement for complex expression methods or specialized strains.Open in a separate windowFig. 1.Chemical structures of iron protoporphyrin IX (FePPIX or heme b), cobalt protoporphyrin IX (CoPPIX), and free base protoporphyrin IX (H₂PPIX).     

Synthesis,characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by ¹H NMR, UV–vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2 mW cm⁻²) of red light (643 nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC₅₀ values. The obtained IC₅₀ values for 5 were 1.4 μM at 15 min irradiation time and 0.7 μM when the time of irradiation was 30 min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.