Using flue gas desulfurization gypsum to remove dissolved phosphorus from agricultural drainage waters.

High levels of accumulated phosphorus (P) in soils of the Delmarva Peninsula are a major source of dissolved P entering drainage ditches that empty into the Chesapeake Bay. The objective of this study was to design, construct, and monitor a within-ditch filter to remove dissolved P, thereby protecting receiving waters against P losses from upstream areas. In April 2007, 110 Mg of flue gas desulfurization (FGD) gypsum, a low-cost coal combustion product, was used as the reactive ingredient in a ditch filter. The ditch filter was monitored from 2007 to 2010, during which time 29 storm-induced flow events were characterized. For storm-induced flow, the event mean concentration efficiency for total dissolved P (TDP) removal for water passing through the gypsum bed was 73 ± 27% confidence interval (α = 0.05). The removal efficiency for storm-induced flow by the summation of load method was 65 ± 27% confidence interval (α = 0.05). Although chemically effective, the maximum observed hydraulic conductivity of FGD gypsum was 4 L s(-1), but it decreased over time to <1 L s(-1). When bypass flow and base flow were taken into consideration, the ditch filter removed approximately 22% of the TDP load over the 3.6-yr monitoring period. Due to maintenance and clean-out requirements, we conclude that ditch filtration using FGD gypsum is not practical at a farm scale. However, we propose an alternate design consisting of FGD gypsum-filled trenches parallel to the ditch to intercept and treat groundwater before it enters the ditch.

Fluorescence absorbance inner-filter decomposition: the role of emission shape on estimates of free Ca(2+) using Rhod-2.

A method for decomposing complex emission spectra by correcting for known inner-filter effects is described. This approach builds on previous work using a linear combination of model emission spectra and combines the known absorption characteristics of the system to fit the composite emission spectrum. Rhod-2, which has a small Stokes shift and significant self-absorption, was used as the model system. By adding the absorption characteristics of Rhod-2 to the model, the degree of fit was significantly improved, thus minimizing residuals, and accurately predicted the spectral shape changes with increasing concentration, [Rhod-2]. More complex studies were conducted with Rhod-2 in isolated cardiac mitochondria with multiple emission and absorption elements. By including known absorbances to the spectral decomposition, the overall precision increased almost four fold. Moreover, this approach eliminated the significant [Rhod-2] dependence on the apparent K(50) and therefore improved the accuracy of free [Ca(2+)] calculations. These data demonstrate that secondary inner-filter correction can significantly improve spectral decomposition of complex emission spectra, which are used in a variety of biological applications.

Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions.

The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.

Interconversions among four M-intermediates in the bacteriorhodopsin photocycle.

Halobacterium salinarum displays four distinct kinetic forms of M-intermediate in its bacteriorhodopsin photocycle. In wild-type, there are mainly two species with time constants near 2 and 5 ms. Under various kinds of stress, two other species arise with time constants near 10 and 70 ms. We show that these four species are interconvertible. Increases in membrane hydrophobicity convert the slower to faster forms. Perturbations caused by Triton X-100 or mutations convert faster to slower forms. The fastest form requires a hydrophobic membrane environment near a ring of four charged aspartate residues in the trimer, namely Asp36, Asp38, Asp102, and Asp104 in the cytoplasmic loop regions. Interconversions of the 2-ms and 5-ms species of the wild-type are accomplished by pH-changes. The potential significance of these findings is discussed.

Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate.

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both deltaGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.

Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro.

The role of Ca(2+) as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca(2+) sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa(3) ratio. The half-maximal concentration (K(1/2)) of SR ATPase was 335 nM Ca(2+). The ATP synthesis dependence was similar with a K(1/2) of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca(2+) increased thapsigargin-sensitive ATP production (maximum approximately 5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca(2+) concentrations (0 to approximately 500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca(2+) that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca(2+) is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.