Pheophytinization

Demidov A A and Ivanov I G 1989 Laser fluorometrio method for measuring the oonoentrations of ohiorophyll a and pheophytin a in aoetone solutions Biol. Bull. Acad. Sc/. USSR 16 228-34... 

This pair of chlorophyll molecules, which as we shall see accepts photons and thereby excites electrons, is close to the membrane surface on the periplasmic side. At the other side of the membrane the symmetry axis passes through the Fe atom. The remaining pigments are symmetrically arranged on each side of the symmetry axis (Figure 12.15). Two bacteriochlorophyll molecules, the accessory chlorophylls, make hydrophobic contacts with the special pair of chlorophylls on one side and with the pheophytin molecules on the other side. Both the accessory chlorophyll molecules and the pheophytin molecules are bound between transmembrane helices from both subunits in pockets lined by hydrophobic residues from the transmembrane helices (Figure 12.16). 

The functional reaction center contains two quinone molecules. One of these, Qb (Figure 12.15), is loosely bound and can be lost during purification. The reason for the difference in the strength of binding between Qa and Qb is unknown, but as we will see later, it probably reflects a functional asymmetry in the molecule as a whole. Qa is positioned between the Fe atom and one of the pheophytin molecules (Figure 12.15). The polar-head group is outside the membrane, bound to a loop region, whereas the hydrophobic tail is... 

Figure 12.16 View of the reaction center perpendicular to the membrane illustrating that the pigments are bound between the transmembrane helices. The five transmembrane-spanning a helices of the L (yellow) and the M (red) subunits are shown as well as the chlorophyll (green) and pheophytin (blue) molecules.

In the bacterial reaction center the photons are absorbed by the special pair of chlorophyll molecules on the periplasmic side of the membrane (see Figure 12.14). Spectroscopic measurements have shown that when a photon is absorbed by the special pair of chlorophylls, an electron is moved from the special pair to one of the pheophytin molecules. The close association and the parallel orientation of the chlorophyll ring systems in the special pair facilitates the excitation of an electron so that it is easily released. This process is very fast it occurs within 2 picoseconds. From the pheophytin the electron moves to a molecule of quinone, Qa, in a slower process that takes about 200 picoseconds. The electron then passes through the protein, to the second quinone molecule, Qb. This is a comparatively slow process, taking about 100 microseconds. 

One apparent discrepancy between the spectroscopic data and the crystal structure is that no spectroscopic signal has been measured for participation of the accessory chlorophyll molecule Ba in the electron transfer process. However, as seen in Figure 12.15, this chlorophyll molecule is between the special pair and the pheophytin molecule and provides an obvious link for electron transfer in two steps from the special pair through Ba to the pheophytin. This discrepancy has prompted recent, very rapid measurements of the electron transfer steps, still without any signal from Ba- This means either... 

FIGURE 22.19 The molecular architecture of PSII. The core of the PSII complex consists of the two polypeptides (D1 and D2) that bind P680, pheophytin (Pheo), and the quinones, Qb- Additional components of this complex include cytochrome -6559,... 

The early stages of catabolism correspond to the replacement of Mg by two H atoms under acidic conditions and/or by the action of Mg-dechelatase and the cleavage of the phytol chain by the enzyme chlorophyllase. The still greenish intermediates are pheophytins, chlorophyUides, and pheophorbides with intact tet-rapyrrole rings. - ... 

Not-senescent and fresh-cut plants are almost devoid of degradation products like pheophytins and pheophorbides because chlorophylls associated with caro-... 

Studies of chlorophyll degradation in heated broccoli juices over the 80 to 120°C range revealed that chlorophylls degrade first to their respective pheophytins and then to other degradation products in what can therefore be described as a two-step process. Both chlorophyll and pheophytin conversions followed a first-order kinetics, but chlorophyll a was more heat sensitive and degraded at a rate approximately twice that of chlorophyll This feature had been observed by other authors. Temperature dependence of the degradation rate could adequately be described by the Arrhenius equation. ... 

Traditionally, dried or powdered plant material is used and extracts can be obtained by mixing the material with food-grade solvents like dichloromethane or acetone followed by washing, concentration, and solvent removal. The result is an oily product that may contain variable amounts of pheophytins and other chlorophyll degradation compounds usually accompanied by lipid-soluble substances like carotenoids (mainly lutein), carotenes, fats, waxes, and phospholipids, depending on the raw material and extraction techniques employed. This product is usually marketed as pheophytin after standardization with vegetable oils. 

Turkmen, N. et al.. Effects of cooking methods on chlorophylls, pheophytins and colour of selected green vegetables, hit. J. Food Sci. TechnoL, 41, 281, 2006. 

In the past, no snitable analytical methodologies were capable of investigating these multiple reactions and even today, the complete extraction and analysis of all the componnds is still a difficult task. The methods for extraction must be optimized for each sample according to the solubility of either phytylated (chlorophylls and pheophytins) or dephytylated (chlorophyllides and pheophorbides) derivatives, often requiring several repeated steps and the use of a single or a mixture of organic solvents. 

Despite being a fast and relatively low-cost method, thin layer chromatography shows low resolution as it involves a large surface in contact with air, promoting photoxidation. Acidity of silica gels should be neutralized because it may trigger chlorophyll degradation by pheophytinization. ... 

As pheophytin a and pheophytin b are the major degradation derivatives formed during extraction, food processing, and storage, some authors reconunend converting chlorophylls into the more stable pheophytins by treatment with HCl, ion exchange resin, or oxalic acid to estimate the chlorophyll contents. ... 

Usually, HPLC analysis resolves four peaks identified by co-chromatography with authentic standards as copper pheophorbide a, Cn(II) chlorin e6, Cn(II) chlorin e4, Cu rhodin g7, and their degradation products, but a sum of other colored components can also be found, for example, native chlorophylls, pheophytins, pheophor-bides, and rodochlorins (free carboxyl forms of pheophorbides) besides epimers, allomers, and degradation products that have been only tentatively identified. 

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