Unlabelled Water Research Articles

RBS and NRA analysis for films with high growth rate prepared by atomic layer deposition

We present the design and operation of a specialized Atomic Layer Deposition (ALD) system, dedicated to stable isotopic tracing experiments of oxide film growth, using isotopically labelled water as the oxide reactant. A small chamber volume allows operation with only very small quantities of water vapor, minimising the consumption of the isotopically labelled water. The first results for growth of ZnO and TiO2 using Diethlyzinc (DEZ) and Tetrakis(dimethylamino)titanium (TDMAT) as the zinc and titanium precursors, and unlabelled water as reactant, are presented, to establish the growth conditions for stoichiometric ZnO and TiO2 on silicon. Absolute film compositions and thickness are determined by RBS and NRA as a function of vapor pulse duration, number of ALD cycles and substrate temperature. Physical thickness is determined by ellipsometry. The first results obtained for growth of ZnO using water highly enriched in 18O are also presented.

Fate of the 15n-labelled faeces fraction of dairy farm effluent (DFE) irrigated onto soils under different water regimes

Irrigation of dairy farm effluent (DFE) onto pasture is the preferred treatment method in New Zealand for this very dilute organic effluent. Whereas the dynamics of the urine fraction is comparatively well understood, there is a lack of data on the fate of the mainly organic faecal fraction. To improve our understanding of the complex turnover processes, we labelled both the inorganic and organic N compounds of the faecal fraction of DFE with 15N. We then measured the 15N dynamics in various soil and plant fractions in a laboratory experiment at two water contents for up to 254 days. Feeding a dairy cow with 15N-labelled pasture yielded faeces that had a mean 15N abundance of 2.95 atom%. Unlabelled urine and water were added to the labelled faeces to construct the DFE, which contained 90.4% of the 15N in the organic nitrogen fraction (2.87 atom%) and 9.6% in the ammonium fraction (1.23 atom%). As N turnover and losses depend on the method of application and the soil structure, we simulated field conditions by surface-applying our DFE onto intact soil cores with pasture. Two soil water treatments were imposed; dry (30% water content) and wet (water table at 17 cm below the soil surface). The surface application resulted in filtration of the DFE, with a high proportion of the 15N remaining on the soil surface, where it was relatively unavailable for plant uptake but prone to gaseous and physical losses. Of the applied 15N, 9.9% in the dry and 13.5% in the wet treatment were still recovered as DFE on the soil surface at day 254. Plant uptake of faecal 15N accounted for 9.3% and 13.0% in the dry and wet treatments, respectively. The bulk of the 15N was recovered in the soil organic nitrogen fraction (35.1% in dry, 42.5% in wet), whereas 15N in inorganic and microbial nitrogen accounted for only very small amounts (< 2%). Total recoveries of the applied 15N in plant, soil and DFE remaining on the surface at day 254 were 58.4% in the dry, and 71.5% in the wet treatment. Separate analysis of the total and ammonium nitrogen contents and 15N enrichments of the constructed DFE and filtered subsamples (0.5 mm, 0.2 μm) showed that the faecal fraction was not labelled homogeneously. Due to this heterogeneity, which was exacerbated by the filtration of DFE on the soil surface, it was difficult to calculate the turnover of the total faecal fraction based on 15N results.

The derivation of the formyl-group oxygen of chlorophyll b in higher plants from molecular oxygen. Achievement of high enrichment of the 7-formyl-group oxygen from 18O2 in greening maize leaves.

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H2(18)O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-7(1)-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H2(18)O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13(1)-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599-602]. The incorporation of isotope led to as much as 77% enrichment of the 13(1)-oxo group of chlorophyll a: assuming identical incorporation into the 13(1) oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The high enrichment using 18O2, and the absence of labelling by H2(18)O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.

Phosphorus Flux through Epilimnetic Zooplankton from Lake Ontario: Relationship with Body Size and Significance to Phytoplankton

The flux of phosphate through epilimnetic zooplankton from Lake Ontario was measured by adding 32PO4 into whole lakewater, monitoring its accumulation by zooplankton over 48 h, and then measuring its release when animals were returned to unlabelled lake water. Phosphorus uptake was calculated as the sum of accumulation and release rates. The 10 species examined ranged in size from 36-μm Codonella cratera to 1-mm Daphnia retrocurva. Phosphorus uptake rates of herbivorous zooplankton ranged about 200-fold and increased with body size both within and among species, although not proportionally. Among species, there was a pronounced allometry, with smaller species having much higher rates per unit size. This allometry is similar to that described for other metabolic parameters and body size. There was no tendency for smaller species to release a greater fraction of the label they took up. These results suggest that the biomass of zooplankton consuming a given phytoplankton production will be much smaller if small zooplankton dominate, and therefore, this phosphorus sink will be much smaller. Further, the flow of phosphorus to higher trophic levels may be reduced. The result of changing the zooplankton size-distribution may be to change the amount of spring total phosphorus available to summer phytoplankton.

Studies of enzyme-mediated reactions. Part 14. Stereochemical course of the formation of cadaverine by decarboxylation of (2S)-lysine with lysine decarboxylase (E.C. 4.1.1.18) from Bacillus cadaveris

The decarboxylation of (2S)-lysine by lysine decarboxylase (E.C. 4.1.1.18) from B. cadaveris is shown to be stereospecific and to proceed with retention of configuration by incubation of [2-3H,U-14C]lysine in unlabelled water and [U-14C]lysine in tritiated water to give labelled samples of [1-3H,U-14C]cadaverine (1,5-diaminopentane) which are chiral by isotopic substitution. The configuration of each diamine is assayed by deamination with the diamine oxidase from pea seedlings (E.C. 1.4.3.6) which stereospecifically removes the hydrogen from the Si-position of the tritiated methylene group.

Studies of enzyme-mediated reactions. Part 12. Stereochemical course of the decarboxylation of (2S)-tyrosine to tyramine by microbial, mammalian, and plant systems.

Tyrosine is proved to be decarboxylated with retention of configuration by three aromatic amino-acid decarboxylases of bacterial, mammalian, and plant origin by experiments based on the synthesis of (αS)-[α-3H]tyrosine and the (αR)- and (αS)-isomers of [α-3H1]tyramine. Incubation of the labelled tyramines with the diamine oxidase from pea seedlings and the monoamine oxidase from rat liver shows that the former enzyme removes Hsi and the latter HRe. These amine oxidases are used to assay the chirality of the [α-3H1]tyramines produced by incubation of suitably labelled tyrosines in tritiated water or in unlabelled water with the partially purified decarboxylases from Streptococcus faecalis and hog kidney. The plant enzyme was studied by administration of (αS)-[α-3H,U-14C]tyrosine to intact Papaver somniferum plants followed by isolation of the resultant papaverine, which retained no tritium; morphine was also isolated to act as an internal standard.

Carbonyl oxygen exchange of glycol monoesters. Rate and equilibrium constants for the formation of a tetrahedral intermediate

Kinetic investigations of the hydrolysis of the 2-phenyl-4,4,5,5-tetramethyl-1,3-dioxolenium ion and 2-phenyl-2-methoxy-4,4,5,5-tetramethyl-1,3-dioxolane furnish rate constants for all three reaction stages of the ortho ester hydrolysis: (1) generation of the dioxolenium ion, (2) hydration of this ion to form hydrogen ortho ester, and (3) breakdown of this species to pinacol monobenzoate. The equilibrium constant for stage (2) can also be obtained. This study complements a previous investigation of 2-phenyl-2-alkoxy-1,3-dioxolanes where similar information was obtained.The rate constants for carbonyl oxygen exchange of the ester products of these reactions, pinacol monobenzoate and ethylene glycol monobenzoate, have been measured. This reaction is shown to proceed by a different mechanism to that normally associated with exchange of carboxylic acid derivatives: cyclization of the glycol monoester to form hydrogen ortho ester, followed by loss of the labelled exocyclic OH group to give 1,3-dioxolenium ion. Reversal of these steps, initiated by an unlabelled water molecule, results in exchange. The relationship of this mechanism with that of the ortho ester hydrolysis is obvious; it is shown that the exchange provides rate constants for the reverse of stage (3). This means that both the forward and reverse rates of this process have been obtained, and this provides the equilibrium constant.

The nature of the proton transfer from an acid group at the active site of an enzyme, to solvent water. The extent of 2H and 3H transfer in the reaction catalysed by triose phosphate isomerase

The extent of transfer of a 2H and of a 3H label in 1-R-[2H or 3H]-dihydroxyacetone phosphate to the 2-position of D-glyceraldehyde 3-phosphate in the reaction catalysed by triose phosphate isomerase has been determined. Since the enzymic base that abstracts the substrate's isotopic label is a carboxylate group, this enzyme-substrate system effectively provides —COO2H and —COO3H in a solution of 1H2O, and allows an investigation of the transfer of 2H and 3H from the carboxyl group to unlabelled water. From the value of the fractionation factor for this proton transfer, it is evident that the mechanism of exchange of isotope on —COOL with the protons of the solvent does not involve a transition state in which L is in flight.

Feeding on dissolved organic matter by some marine animals

The uptake of dissolved organic matter (DOM) by various marine invertebrates was determined using 14C-labelled algal hydrolysate and compared with the uptake of particulate (algae and bacteria) organic matter. Uptake was maximal in those forms having a large active epithelial surface exposed to the aqueous medium. Enteropneust and echinoderm larvae, bivalve molluscs, pteropods, sponges, coelenterate medusae and chaetognaths (in descending order) all exhibited significant capacity for uptake (10–40% of the total metabolic requirements) when exposed to 0.50 mg C/1 of DOM ∗ ∗ See explanation of symbols with equation (1). . Animals with chitinous exoskeletons (various crustaceans) had a considerably reduced capacity, DOM accounting for 2–5% of their total intake of organic matter. Uptake, at least in certain forms (bivalves, pteropods), bears a relationship with the environmental DOM concentration. That part of the DOM taken up by the animals entered into their metabolic pool could be demonstrated by the recovery of labelled CO 2 released by the animals for a considerable period after their return to unlabelled water. The importance of this phenomenon in the nutrition of marine animals and the need to consider it as a significant factor in evaluating energy flow in the ecosystem are emphasized.

Further Evidence for the Relative Immobility of Water in Sieve Tubes of Willow

AbstractGradients of tritiated water, 35S‐sulphate and 32P‐phosphate, were established in isolated segments of willow stems. Sieve tube exudate was collected as honeydew from the high activity end of the segment. After girdling the stem a few centimetres from the site of sieve tube puncture, the specific activity of 35S and 32P in the honeydew rose, whilst the specific activity of tritium remained constant.These findings indicate that prior to girdling, unlabelled sulphates and phosphates were contributing to the honeydew, whilst there had been no detectable contribution by unlabelled water from the low activity end of the segment.The data support the conclusions drawn from previous experiments by Peel et al. (1969), that water is relatively immobile in sieve tubes of willow when compared to solutes.