Tuning fast excited-state decay by ligand attachment in isolated chlorophyll a.

Excited-state dynamics plays a key role for light harvesting and energy transport in photosynthetic proteins but it is nontrivial to separate the intrinsic photophysics of the light-absorbers (chlorophylls) from interactions with the protein matrix. Here we study chlorophyll a (4-coordinate complex) and axially ligated chlorophyll a (5-coordinate complex) isolated in vacuo applying mass spectrometry to shed light on the intrinsic dynamics in the absence of nearby chlorophylls, carotenoids, amino acids, and water molecules. The 4-coordinate complexes are tagged by quaternary ammonium ions while the charge is provided by a formate ligand in the case of 5-coordinate complexes. Regardless of excitation to the Soret band or the Q band, a fast ps decay is observed, which is ascribed to the decay of the lowest excited singlet state either by intersystem crossing (ISC) to nearby triplet states or by excited-state relaxation on the excited-state potential-energy surface. The lifetime of the first excited state is 15 ps with Mg2+ at the chlorophyll center, but only 1.7 ps when formate is attached to Mg2+. When the Soret band is excited, an initial sup-ps relaxation is observed which is ascribed to fast internal conversion to the first excited state. With respect to ISC, two factors seem to play a role for the reduced lifetime of the formate-chlorophyll complex: (i) The Mg ion is pulled out of the porphyrin plane thus reducing the symmetry of the chromophore, and (ii) the first excited state (Q band) and T3 are tuned almost into resonance by the ligand, which increases the singlet-triplet mixing.

Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon's assay.

A recent publication (Esteban in New Phytol 217:341-342, 2018) describes how the use and citation of the assay of chlorophylls a and b extracted in aqueous 80% acetone by Arnon (Plant Physiol 2:1-15, 1949) is increasing, even in journals with high impact factors. This is a very disconcerting situation: the assay is outdated because it relies on the seriously under-estimated extinction coefficients of Mackinney (J Biol Chem 140:315-322, 1941), and the assay of chlorophylls is one of the most important, and much reported, procedures in studies of photosynthesis and related plant biological fields. Using the assay has led to the accumulation of masses of inaccurate data and confusion during the resolution of some plant biological problems. A summary not only of an accurate assay of chlorophylls in aqueous 80% acetone but also of a long-known method to correct the data obtained by Arnon's procedure (cf. Porra et al. in Biochim Biophys Acta 975:384-394, 1989) is briefly described below together with references to reliable assays in this and other solvents by other authors.

Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f.

Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance maxima in the blue (Soret) and red (Qy) regions with Chl b, Chl d, and Chl f each possessing a single formyl group at a unique position. Relative to Chl a the Qy absorbance maximum of Chl b is blue-shifted while Chl d and Chl f are red-shifted with the shifts attributable to the relative positions of the formyl substitutions. Reduction of a formyl group of Chl b to form 7-hydroxymethyl Chl a, or oxidation of the vinyl group of Chl a into a formyl group to form Chl d was achieved using sodium borohydride (NaBH4) or ß-mercaptoethanol (BME/O2), respectively. During the consecutive reactions of Chl b and Chl f using a three-step procedure (1. NaBH4, 2. BME/O2, and 3. NaBH4) two new 7-hydroxymethyl Chl a species were prepared possessing the 3-formyl or 3-hydroxymethyl groups and three new 2-hydroxymethyl Chl a species possessing the 3-vinyl, 3-formyl, or 3-hydroxymethyl groups, respectively. Identification of the spectral properties of 2-hydroxymethyl Chl a may be biologically significant for deducing the latter stages of Chl f biosynthesis if the mechanism parallels Chl b biosynthesis. The spectral features and chromatographic properties of these modified Chls are important for identifying potential intermediates in the biosynthesis of Chls such as Chl f and Chl d and for identification of any new Chls in nature.

Biochemical, Micronutrient and Physicochemical Properties of the Dried Red Seaweeds Gracilaria edulis and Gracilaria corticata.

The present study sought to evaluate the nutritional composition and physicochemical properties of two dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis. Proximate composition of the dried seaweeds revealed a higher content in carbohydrates (8.30 g/100 g), total crude protein (22.84 g/100 g) and lipid content (7.07 g/100 g) in G. corticata than in G. edulis. Fatty acids profile showed that G. corticata samples contain higher concentrations of saturated fatty acids, such as palmitic and stearic acids, and polyunsaturated ones such as α-linolenic and docosahexaenoic acids. Contrariwise, G. edulis contained higher amounts of monounsaturated oleic acid. Total amino acid content was 76.60 mg/g in G. corticata and 65.42 mg/g in G. edulis, being the essential amino acid content higher in G. edulis (35.55 mg/g) than in G. corticata (22.76 mg/g). Chlorophyll a was found in significantly higher amounts in G. edulis (17.14 µg/g) than G. corticata, whereas carotenoid content was significantly higher in G. corticata (12.98 µg/g) than in G. edulis. With respect to physical properties, both water- and oil-holding capacities were similar in both seaweeds, whereas swelling capacity was higher in G. edulis. In view of the results, the present study suggests that G. corticata and G. edulis contains important nutrients for human health and are possible natural functional foods.

Cremophor EL Nano-Emulsion Monomerizes Chlorophyll a in Water Medium.

In this paper, the application of a non-ionic detergent Cremophor EL for monomerization of chlorophyll a in an aqueous medium is studied. The spectrophotometric properties of chlorophyll a encapsulated into the Cremophor EL nano-emulsion system were characterized by electronic absorption, steady-state and time-resolved fluorescence as well as circular dichroism spectroscopy. The results have shown that chlorophyll a dissolves more efficiently in the aqueous medium containing low-level Cremophor (5 wt%) than at an ethanolic solution even in the concentration of 10-4 M. The molecular organization of the chlorophyll a in the Cremophor EL nano-micelles was also investigated by means of Raman spectroscopy. The spectral changes in the frequency of the C=O stretching group were used to distinguish the aggregation state of chlorophyll. It was revealed that chlorophyll a exists dominantly in the monomeric form in the Cremophor EL aqueous solution. The promising aspect of the use of Cremophor EL nano-emulsion as a delivery system is to maintain stable chlorophyll monomer in an aqueous medium. It would open the potential for a new, practical application of chlorophyll a in medicine, as a dietary supplement or studies on molecular organization of chlorophyll a in the well-defined artificial system.

Identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale using ultrasound-assisted extraction.

The identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale was determined. The ultrasound-assisted extraction was utilized for obtaining the chlorophyll compounds from biomass. A total of eleven molecules were separated in microalgae chlorophyll extract, with pheophytin a' (371µg·g-1) and chlorophyll a (159.3µg·g-1) as the major ones. The chlorophyll extract was shown to be a potent scavenger of peroxyl radical, being almost 200 times more potent than α-tocopherol. These facts suggest the microalgae Phormidium autumnale as potential source of bioactive tetrapyrrole compounds.

Anti-inflammatory effects of dulse (Palmaria palmata) resulting from the simultaneous water-extraction of phycobiliproteins and chlorophyll a.

The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin ß-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse.