Determination of photosynthetic pigments

UNESCO,1966. Determination of photosynthetic pigments in sea-water. Monographs on Oceanographic Methodology, Vol.l, pp. 1-69. 

Rai, H., 1973. Methods involving the determination of photosynthetic pigments using spectrophotometry. Verh. Int. Ver. Theor. Angew. Limnol., 18 1864—1875. 

For comparison with other plant samples, take 0.05 or 0.1 ml of this concentrated pigment extract, dilute to 5 ml with 100% acetone or diethyl ether, and determine the amount of photosynthetic pigments using the absorption coefficients and equations given in UNITF4.3. 

The analysis of carotenoid identity, conformation, and binding in vivo should allow further progress to be made in understanding of the functions of these pigments in the photosynthetic machinery. One of the obvious steps toward improvement could be the use of continuously tuneable laser systems in order to obtain more detailed resonance Raman excitation profiles (Sashima et al 2000). This technique will be suitable for the investigation of in vivo systems with more complex carotenoid composition. In addition, this method may be applied for the determination of the energy of forbidden Sj or 2 Ag transition. This is an important parameter, since it allows an assessment of the energy transfer relationship between the carotenoids and chlorophylls within the antenna complex. 

Three main tendencies have been underlined in recent studies of structure and action mechanism ofbacterial photosynthetic reaction centers. The crystallographic structure of the reaction centers from Rps. viridis and Rb. spheroids was initially determined to be 2.8 and 3 A resolutions (Michel and Deisenhofer et al., 1985 Allen et al., 1986). Resolution and refinement of these structures have been subsequently extended to 2.2, 2.3 and 2.6 A. (Rees et al., 1989 Stowell et al., 1997, Fyfe and Johns, 2000 Rutherford and Faller, 2001). Investigations of the electronic structure of donor and acceptor centers in the ground and exited states by modern physical methods with a combination ofpico-and femtosecond kinetic techniques have become more precise and elaborate. Extensive experimental and theoretical investigations on the role of orbital overlap and protein dynamics in the processes of electron and proton transfer have been done. All the above-mentioned research directions are accompanied by extensive use of methods of sit-directed mutagenesis and substitution of native pigments for artificial compounds of different redox potential. 

Photosynthetic pigments, chlorophylls, and carotenoids have a clear hydro-phobic character and are usually analyzed by C18-reversed-phase (RP) columns. A C30-RP appeared on the market. The C30-RP is particularly efficient in the separation of carotenoids because the interactions of the pigments and the stationary phase are maximized by the similar size. With this phase, many cis isomers of the same carotenoid are separated from each other (Emenhiser et al., 1995 Lacker et al., 1999 Sharpless et al., 1996). This C30-RP has been successfully applied to the determination of saponified carotenoids in orange juice (Rousseff et al., 1996). However, when a mixture is complex, coelutions may become rapidly limiting and less selective stationary phases, such as the C18-RP, are therefore preferably... 

The compounds included in this section have mainly been detected in the particulate fractions of seawater and are su ested to correlate with biological activity. The oldest determined compounds in this class are the photosynthetic pigments, mainly the chlorophylls. The methods for the determination of the plant pigments have been standardised by international agreements (UNESCO, 1966) and the published methods of Strickland and Parsons (1972). A review of the methods for the determination of both total and individual p ments has been presented by Rai (1973). 

In the next section we take a quick look at the triplet state and discuss the origin and significance of the ZFS. This is followed by an outline of some of the ODMR experiments that are in common use to determine static and dynamic triplet state properties in biopolymers. After a description of experimental equipment and methods used in ODMR spectroscopy, we conclude with some examples that illustrate the wealth of information provided by the application of ODMR to the study of tryptophan triplet states in proteins. Because this chapter is intended to focus on methods, we present neither a historical development of ODMR spectroscopy, nor an extensive review of its application to biopolymer studies. We refer the interested reader, instead, to some early reviews on the general methods and results of ODMR spectroscopy, as well as to a book and to additional reviews that deal more specifically with biological applications of ODMR. Applications to photosynthetic pigments and the reaction centers, in particular, have been reviewed thoroughly by Hoff. - ... 

Our knowledge of the stereochemistry of porphyrins and related tetrapyrrole macrocycles has expanded rapidly since the first reported x-ray structure determination in 1959 The structures of metallotetrapyrrole complexes are of interest because of the common occurrence of this type of macrocycle in biological systems. As is well known, foremost among these are the heme proteins (iron derivatives), the various photosynthetic pigments (magnesium complexes), the vitamin Bn coenzyme (cobalt corrinoids), and coenzyme F430 (nickel corphinoids) of the methanogenic bacteria. 

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