Chlorophyll assay

At a fixed dose-rate (45 5 pphm), the time of exposure was varied from zero to 90 min, and treated plants incubated a further 24 hr before chlorophyll assay. This test also revealed non-linear responses (Table I) for each organ on the bases of chlorophyll and fresh weight contents using seedlings of sensitive ages. 

Seedlings of the given ages were treated at 45 5 pphm for the lengths of time given and incubated a further 24 hr before fresh weight and chlorophyll assays. 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

Ferruzzi, M.G. et al.. Antioxidant and antimutagenic activity of dietary chlorophyll derivatives determined by radical scavenging and bacterial reverse mutagenesis assays, J. Food ScL, 67, 2589, 2002. 

The research into the biological activities of chlorophylls developed over the past 20 years is also important although very few in vivo assays concerning then-potential health benefits have been performed. Far fewer studies have focused on chlorophylls in comparison to carotenoids. Efforts to stabilize chlorophylls in pro-... 

Sodium copper chlorophyllin, approved by the FDA as a color additive in citrus-based dry beverage mixes, should have a ratio of absorbance (SoretQ band) not less than 3.4 and not more than 3.9. In Europe, purity criteria of the food additives E141[i] and E141[ii], which are copper complexes of chlorophyll and chlorophyllin, respectively, are set out in the EC color specifications that include identification and spectrophotometric assay tests. ... 

Porra, R.J., Thompson, W.A., and Kriedemann, P.E., 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, Biochim. Biophys. Acta, 975,384, 1989. 

A miniaturized version of the conventional flask method with S. capricomutum has been developed by Blaise et al [136]. In this assay the algae are exposed to the toxicant in 96-well microplates for a period of 96 hours, after which the cell density is determined using a hemocytometer or electronic particle counter. ATP content measurements [136] or chlorophyll fluorescence [141,142] have also been proposed as test criteria. Compared to the flask method, the main advantages of the microplate assay are (a) the small sample volumes and reduced... 

Next, the studies were extended to evaluate the effect of the compounds on duckweed which is one of the best characterized models for assessing phytotoxic activity. The duckweed assay system makes it possible to study the toxic effects throughout the plant life cycle, as well as to plant specific toxic effects which target photosynthesis. Of all the isolates, only 12 and 13 showed significant phytotoxicity on duckweed at concentrations of 100 xM and 200 xM inhibiting plant growth by 80% and 100%, respectively and chlorophyll production by 40% and 84%, respectively. ... 

Figure 1. Chlorophyll content and fresh weight of the cucumber cotyledon pair. O == untreated plants, X = 4 hrs at 24 2 pphm ozone and held 24 hrs before assay. Vertical bars indicate standard direction of the mean.

Figure 4. Chlorophyll content and fresh weight of the soybean first-leaf pair. Legend as in Figure 1. Plants treated for 4 hrs at SO 5 pphm were assayed 24 and 48 hrs later.

Several factors affect the salt sensitivity of plants. We first evaluated the effect of light intensity on the salt-stress. Figure 2 clearly showed that tobacco seedlings were more severely affected by salt stress under high light intensity both on the basis of chlorophyll content and fresh wei t increase. This result indicated that light, probably photosynthetic process, would be involved in the salt-stress. To characterize the salt-stress mechanism, we examine the effect of salt on the photosynthetic activities of isolated thylakoid membranes. Previously, we reported that the presence of salt in assay inhibited the photosystem II activity of tobacco thylakoid membranes but not photosystem I activities (Murota et al., 1994). Then, we further examined the effect of salt on the irreversible photodamage of thylakoid membrane activity. 

Malkin used two methods to assay the photochemical activity of both the intact PS 1-200 complex and the complex that had lost one phylloquinone. One method measured oxygen uptake by using methyl violo-gen as the acceptor photoreduced by photosystem I, supported by plastocyanin and reduced DCIP as the electron donors. The other method measured the amount of FeS-A photoreduced at 15 K by EPR spectroscopy. It turned out that both the extracted and unextracted samples gave nearly identical results, namely -500 p,M Oj/mg Chl /i, and the EPR spectra showed nearly identical amounts of FeS-A reduced on an equivalent chlorophyll basis. These results thus indicated that there is one nonfunctional phylloquinone that is easily removed by extraction with an organic solvent and one tightly bound, functional phylloquinone that is presumably present in a highly hydrophobic environment and which was subsequently identified as an intermediary electron carrier in the acceptor chain of photosystem I. 

Before running the acquisition on a flow cytometer, we usually calibrate the machine using the three-color BD CaliBRITE kit containing unlabeled beads, fluorescein isothiocyanate (PITC)-labeled beads, phycoerythrin (PE) labeled beads, and peridin chlorophyll protein (PerCP)-labeled beads. In addition, we add the allophycocyanin (APC)-labeled beads to setup all four colors used in the assay. This procedure allows determining the correct settings (compensation and detector/amplitude) when several colors are simultaneously used. 

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