Modern methods of plant analysis

Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes

Abstract: Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.

Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy

Abstract: The extinction coefficients for chlorophylls a and b in diethylether (Smith, J.H.C. and Benitez, A. (1955) in Modern Methods of Plant Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143–196, Springer-Verlag, Berlin), used in this paper as primary standards, were verified, to within an error of less than 1%, by magnesium determination using atomic absorbance spectrophotometry. We also report the determination of accurate extinction coefficients for chlorophylls a and b in N,N ′-dimethylformamide, methanol or buffered 80% aqueous acetone. Highly purified chlorophylls were used and methods were employed which not only minimize errors due to evaporation of the volatile solvents employed in their estimation but also eliminate variable micro-contamination by chlorophyll degradation products, a potential source of inconsistency between the extinction coefficients obtained in each of these three solvents. Using these new coefficients, expressed as both millimolar and specific coefficients, we have derived new simultaneous equations to obtain chlorophyll concentrations as nmol/ml and μg/ml, respectively. These equations were applied to data obtained with leaf discs from spinach and Flindersia brayleyana extracted with the three specified solvents and to a concentrated solution (in N,N′ -dimethylformamide) of a chlorophyll a + b mixture added to the threesolvent systems. The validity of these equations is proven by the consistency of the chlorophyll determinations and of the chlorophyll a/b ratios. New simultaneous equations, compatible with the equations derived for the threesolvents, are presented for the assay of chlorophylls a and b converted to their cyclic hydroxylactone derivatives by extraction with alkaline pyridine reagent (2.1 M pyridine in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including the chlorophyll content and chlorophyll a/b ratios of plant thylakoids and chlorophyll-protein complexes, have been obtained in 80% aqueous acetone with the much used simultaneous equations of Arnon (Arnon, D.I. (1949) Plant Physiol. 24, 1–15). For this reason we include conversion factors whichcorrect these earlier data and make it compatible with data calculated with the simultaneous equations presented in this paper. The importance of these corrections to the formulation of meaningful models of the photosynthetic apparatus is demonstrated. Our results also indicate that grinding leaf discs with N,N ′-dimethylformamide is a more reliable method for extracting all chlorophylls than shaking with this solvent for 24 h.

By-products of plant food processing as a source of functional compounds — recent developments

Abstract: There is a rapidly growing body of literature covering the role of plant secondary metabolites in food and their potential effects on human health. Furthermore, consumers are increasingly aware of diet related health problems, therefore demanding natural ingredients which are expected to be safe and health-promoting. By-products of plant food processing represent a major disposal problem for the industry concerned, but they are also promising sources of compounds which may be used because of their favourable technological or nutritional properties. The purpose of this review is to highlight the potential of selected by-products as a source of functional compounds.

The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b

Abstract: Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the Arnon assay [Arnon DI (1949) Plant Physiol 24: 1–15], employed simultaneous equations for determining the concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials. These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early inaccurate spectrophotometric data. Thus, Arnon’s equations give inaccurate chlorophyll a and b determinations and, therefore, inaccurate chlorophyll a/b ratios, which are always low. This paper describes how the ratios are increasingly and alarmingly low as the proportion of chlorophyll a increases. Accurate extinction coefficients for chlorophylls a and b, and the more reliable simultaneous equations derived from them, have been published subsequently by many research groups; these new post-Arnon equations, however, have been ignored by many researchers. This Minireview records the history of the development of accurate simultaneous equations and some difficulties and anomalies arising from the retention of Arnon’s seriously flawed equations.

Allgemeine Charakterisierung eines Enzyms

Abstract: Kann ein physiologischer Vorgang auf eine enzymatische Wirkung zuruckgefuhrt werden (vgl. S. 301), so besteht die folgende Aufgabe darin, Naheres uber die Eigenschaften des beteiligten Enzyms zu ermitteln. Hierzu gehoren die Bestimmung der Reaktions- und Substratspezifitat sowie die Ermittlung der Bedingungen, unter denen eine optimale Wirkung des Enzyms gegeben ist. Wesentlich zur Charakterisierung ist ferner die Untersuchung der Stabilitat des Enzyms und dabei insbesondere die Feststellung, ob es sich um ein Ferment handelt, das zur vollen Aktivitat dialysable Cofaktoren benotigt. Falls diese Frage bejaht wird, ist auch die Bestimmung der unerlaslichen Cofaktoren anzuschliesen. Uberdies bietet auch der Nachweis der Lokalisation des Enzyms in der Zelle (oder im Zellverband) eine entscheidende Moglichkeit zur Charakterisierung des Fermentes. Hinzu kommt schlieslich noch die Untersuchung der Wirkung einzelner Inhibitoren1 auf das Enzym, die zu weitgehender Klarung des Reaktionsmechanismus beitragen kann und eine Abgrenzung der Eigenschaften des untersuchten Fermentes gegenuber ahnlichen Enzymen erlaubt.

Optical Rotatory Dispersion. Its Application to Protein Conformation.

Abstract: Optical rotation has been found to be one of the most convenient methods of following the denaturation of proteins. Generally speaking denaturation can be defined as a process or sequence of processes in which the spatial arrangement of the polypeptide chains within the molecule is changed from that typical of the native protein to a more disordered arrangement (Kauzmann 1959). The terms “configuration”, “conformation” and “state of folding” are widely used for spatial arrangement. It is probably best to follow the suggestion of Blout (1960) and restrict the use of “configuration” to its original sense, i.e. the spatial arrangement around an asymmetric carbon atom, and to use “conformation” for the shape of the molecule in its entirety. The properties discussed in the previous Chapter i.e., viscosity, diffusion, sedimentation, and light scattering — can all furnish information on the overall shape of proteins or other macromolecules and changes in this shape with environment. Thus Doty, Bradbury and Holtzer (1956) were able to show using these methods, together with streaming birefringence, that poly-γ-benzyl-L-glutamate could exist in two conformations, the α-helix and the solvated randomly coiled form, depending on the solvent. The change from α-helix to random coil was accompanied by marked changes in the optical rotatory properties of the polypeptides. It is to be expected that an α-helical structure should contribute to the rotatory power of a polypeptide since helices are asymmetric and not superimposable on their mirror images. The work on polypeptides has shown that rotatory dispersion is capable of providing information on the folding of the polypeptide chain in proteins and the changes accompanying denaturation.