Chlorophyll

Abstract: The chloroplast, as the seat of chlorophyll pigments in plants, occupies a unique position in the economy of the green cell. In recent years there has been a renewed interest in the reactions and properties of chloroplasts as a result of the work of Hill (11, 12) and Hill and Scarisbrick (13, 14) who demonstrated that the reaction characteristic of photosynthesis in green plants, the evolution of oxygen, occurs in appreciable quantities in isolated chloroplasts under the influence of light and in the presence of suitable oxidants (2, 7, 8, 26). In the course of an investigation of oxygen evolution by isolated chloroplasts it was deemed important to explore their enzymatic composition. Of special interest were considered enzymes capable of participating in oxidation-reduction reactions, and more particularly, those localized principally, if not entirely, in the chloroplasts. This paper presents evidence that a copper enzyme, polyphenoloxidase (otherwise known as tyrosinase or catecholase), is localized in the chloroplasts of spinach beet (chard), Beta vu?garis.

Abstract: A photo-induced cyclic peroxidation in isolated chloroplasts is described In an osmotic buffered medium, chloroplasts upon illumination produce malondialdehyde (MDA)—a decomposition product of tri-unsaturated fatty acid hydroperoxides—bleach endogenous chlorophyll, and consume oxygen These processes show ( a ) no reaction in the absence of illumination; ( b ) an initial lag phase upon illumination of 10–20 minutes duration; ( c ) a linear phase in which the rate is proportional to the square root of the light intensity; ( d ) cessation of reaction occurring within 3 minutes after illumination ceases; and ( e ) a termination phase after several hours of illumination The kinetics of the above processes fit a cyclic peroxidation equation with velocity coefficients near those for chemical peroxidation The stoichiometry of MDA/O 2 = 002, and O 2 Chl bleached = 69 correlates well with MDA production efficiency in other biological systems and with the molar ratio of unsaturated fatty acids to chlorophyll The energies of activation for the lag and linear phases are 17 and 0 kcal/mole, respectively, the same as that for autoxidation During the linear phase of oxygen uptake the dependence upon temperature and O 2 concentration indicates that during the reaction, oxygen tension at the site of peroxidation is 100-fold lower than in the aqueous phase It is concluded that isolated chloroplasts upon illumination can undergo a cyclic peroxidation initiated by the light absorbed by chlorophyll Photoperoxidation results in a destruction of the chlorophyll and tri-unsaturated fatty acids of the chloroplast membranes

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.

Abstract: In a previous paper (l), studies were made of chlorophyll degradation products frequently to be found in leaf extracts. The solation of spectroscopically pure chlorophylls a and b was then considered (2). The logical outcome of these studies is the application of the data to the determination of chlorophyll in extracts. It is complicated by the suggestion that chlorophyll as known to Willstatter and Stoll is an artifact. Meyer (3) states that Stall’s chlorophyll preparations cannot account for the absorption of crude leaf extracts, except on the basis of an abnormal 9 : I ratio of chlorophyll a to 6, and even then a discrepancy occurs in the green at about 5400 8. This paper deals with the estimation of chlorophyll in plant extracts by application of absorption coefficients of the isolated solid chlorophyll components. The question of artifacts is thereby automatically clarified. A secondary problem arises in the substantial effect of solvent on the coefficients, and this will be dealt with first. Effect of Solvent on Absorption CoefJicients of Chlorophylls a and b -Absorption coefficients have been reported (2) for the chlorophylls in anhydrous acetone in some detail. For certain comparisons, a few values were also given for benzene and anhydrous et’her ((2) Table III).’ Through the courtesy of Dr. F. I’. Zscheile,