High-performance liquid chromatographic determination of pyrophosphate in the presence of a 20,000-fold excess of orthophosphate.

An HPLC method was based on anion-exchange separation of pyrophosphate (diphosphate) and orthophosphate and postcolumn spectrophotometric detection at 140 degrees C with a molybdenum(V)-molybdenum(VI) reagent. The reagent was easy to prepare, stable for at least 6 months at room temperature, and ready for the determination of pyrophosphate and orthophosphate by the so-called heteropoly blue method without use of any reducing agent. A photodiode-array detector for HPLC indicated the spectral characteristics of the heteropoply blue complex that was detectable at 330-800 nm. The HPLC method had a wide dynamic range from 3 x 10(-7) to 5 x 10(-4) M for both pyrophosphate and orthophosphate with a relative standard deviation of measurement of 10 approximately 2%. Pyrophosphate of 5 x 10(-7) and 5 x 10(-6) M, respectively, could be determined in the presence of a 20,000-fold excess of orthophosphate; 0.01 and 0.1 M.     

Phosphorus availability and alkaline phosphatase activities in two Israeli fishponds

1. Alkaline phosphatase activities and the release of orthophosphate from endogenous substrates by these enzymes were measured in waters from two commercial fishponds in the watershed area of Lake Kinneret (Northern Israel). These data were compared with results from the lake at seasons of adequate or limited phosphorus supply. In the fishponds, high Relative Phosphatase Activity ratios (>2) and relatively large amounts of orthophosphate extracted from plankton by autoclaving (average 27% of readily available phosphorus) indicated adequate, or even excess, levels of phosphorus availability despite elevated pond productivity (2 to 3 tons carp/ha/yr). Therefore, we suggest that decreasing routine phosphorus fertilization of these ponds would not affect overall productivity but would eventually lower the amounts of phosphorus reaching Lake Kinneret.
2. In general, the R. P. A. ratio may be a useful index to evaluate phosphorus availability for a wide range of natural waters. Values for this ratio of <1 and >2 appear indicative of limited or adequate phosphorus availability respectively.
3. Three sources of orthophosphate, (Pi), readily available to phytoplankton, are indicated: (1) enzymatically released Pi, (2) Pi in intracellular pools and (3) Pi initially present in the water. Although the first source is always important, relatively greater amounts of Pi are contributed by the other fractions in situations of plentiful phosphorus availability.
4. Activity of free dissolved phosphatases was found in filtered samples of fishpond water. However, neither these enzymes or added phosphatases released significant amounts of Pi from the dissolved organic phosphorus compounds in the filtered water.

     

Occurrence ofAeromonas hydrophila in limnetic environments: Relationship of the organism to trophic state

The densities ofAeromonas hydrophila in various natural waters were found to be strongly correlated with a relative index proposed for use in trophic state assessments of fresh waters. No such correlation was found with the recoverable heterotrophic population of whichA. hydrophila is a part.A. hydrophila was found to be seasonally distributed with maximal densities occurring during summer through early fall. It was also found to be spatially distributed within a pond with the most consistent densities occurring from 1 m depth down to that depth in the water column where the temperature reaches 16°C. The densities of the organism correlated most strongly with total phosphorus, chlorophylla, and Secchi depth. Moderate correlations were found with dissolved phosphorus, Kjeldahl nitrogen, and dissolved organic carbon. Little or no correlation was obtained with ammonia, orthophosphate, pH, alkalinity, or dissolved oxygen. The discriminating ability that theA. hydrophila density measurements provide in the oligotrophic through mesotrophic range appears to exceed those of presently available methods. The facility and sensitivity of the enumeration method forA. hydrophila should make it a useful tool for trophic state assessments.     

High-performance liquid chromatography for the purification of restriction endonucleases, application to BanII, SacI, and SphI

Conventional fractionation methods are time consuming, thus they prolong the time required to process low-stability restriction enzymes. We now report a rapid and effective two-step chromatographic method that affords high purity endonucleases in a short time. Accordingly, an inexpensive chromatographic adsorbent such as phosphocellulose or dyed agarose in the first step is coupled to a high-performance ion exchanger, namely, MonoQ, in the second step. The purification schemes reported here are now in routine use to prepare high-purity BanII, SacI, and SphI as judged by the "overdigestion" and "cut-ligate-recut" stringent quality tests.     

细菌解磷能力测定方法的研究

选择分解有机磷能力较强的3株细菌和溶解磷矿粉较强的4株细菌,砂培4周后,用不同的方法测定水浸提液中磷的含量。发现不同的细菌解磷能力差异很大,细菌在分解磷化合物的同时,一部分磷被细菌同化,一部分以无机磷酸盐状态贮藏在细菌细胞内。直接测定浸提液中无机磷酸盐的含量,将大大低估细菌的解磷能力,必须将浸提液消煮,才能比较正确地反映细菌分解磷的能力。     

Relevance of a perchloric acid extraction scheme to determine mineral and organic phosphorus in swine slurry

To increase the phosphorus recycling potential from swine slurry, mineral phosphorus products which could be used as fertilizers should be obtained and new processes need to be investigated. A routine method is needed to better evaluate the dissolved and solid mineral phosphorus in swine slurry. Cold perchloric acid extraction method previously developed for wastewater or sludge analysis was adapted. Ionic chromatography was used to measure orthophosphate in extracts. Only one extraction step was needed to distinguish between mineral and organic phosphorus in slurry. Reproducibility of the method was high (less than 5% of variation on the measured fractions). Selectivity was assessed by adding several organic and mineral phosphorus sources in the slurry. Cold perchloric extraction followed by ionic chromatography was very selective in quantifying both the mineral and organic forms of phosphorus in swine slurry.     

Phosphatase activity and its rôle in the mineralization of organic phosphorus in coastal sea water

Alkaline phosphatase activity in sea-water samples taken from Tokyo, Sagami, and Suruga Bays in Japan was measured by a sensitive fluorometric method. There is a relationship between the phosphatase activity and bacterial biomasses in these three bays. The phosphatase-producing bacteria accounted for 40, 46, and 41% of heterotrophic bacteria in Tokyo, Sagami and Suruga Bays, respectively. Significant amounts of phosphatase-hydrolysable organic phosphorus were found in the euphotic zones of these bays and this organic phosphorus fraction accounted for 19 and 50% of organic phosphorus in Tokyo and Sagami Bays, respectively. Phosphatase-hydrolysable organic phosphorus was decomposed completely in the euphotic zone suggesting that this organic fraction is re-cycled in the primary production of the bays. Decomposition of natural organic phosphorus by enzymes was followed by measuring the release of inorganic orthophosphate from samples saturated with chloroform. Release of inorganic phosphorus proceeds rapidly for 2 or 3 days followed by a slow release. Enzymes present in the samples contributed to the decomposition of 50% of the organic phosphorus and the phosphatase enzyme was responsible for the decomposition of one-third of the hydrolysable organic phosphorus in the samples. Potential phosphatase activity in the samples was found to be indicative of the extent and rate of decomposition of organic phosphorus in coastal waters.     

Determination of biologically available phosphorus using a radiobioassay technique

SUMMARY.

• 1 A kinetic approach in which the uptake rate of simultaneous additions of orthophosphate and carrier-free phosphorus- 32 by a standard culture of the green alga Selenastrum capricornutum under near neutral conditions was used to obtain an estimate of the readily available components of the total phosphorus pool in natural freshwater samples.
• 2 A comparison was made between the values of the biologically available phosphorus obtained using this radiobioassay technique and those obtained by the soluble reactive phosphorus procedure for thirty-one freshwater samples. Only about three-quarters of the phosphorus measured as soluble reactive phosphorus was readily available to algae. The results agreed with those obtained in an earlier study in which an enzymatic procedure was used to determine orthophosphate.
• 3 The results of tests carried out on the water samples used in this study were consistent with the conclusion that the presence of a hydrous ferric oxide-orthophosphale complex in the samples caused the soluble reactive phosphorus value to be greater than the corresponding biologically available phosphorus value.

     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Analytical grade C-phycocyanin obtained by a single-step purification process

C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Particulate and dissolved phosphorus forms in freshwater: composition and analysis

In recent research, particulate and dissolved phosphorus components have been separated and characterized on the basis of their physical and chemical properties and partly by their origins.Classical operationally defined monitoring variables (dissolved reactive phosphorus, dissolved unreactive phosphorus and particulate phosphorus) are not congruent with known specific physical or chemical components of phosphorus in natural waters or with their bioavailability.Physical isolation of true particles, colloids and molecules of various sizes is possible at present although it is not recommended for routine use.Chemical characterization of particulate phosphorus is performed mainly by sediment extraction procedures (specialized for inorganic species) and — to a lesser degree — by cell extraction procedures (specialized for organic compounds). The extraction procedures are similar and physical preseparation or alternative procedures (e.g. enzymatic assays) are essential.Smaller colloids and dissolved compounds are physically separated by column chromatography and are often chemically characterized by degradation on the addition of specific enzymes.     

Treatment of dairy wastewater using constructed wetlands and intermittent sand filters

In Ireland, the most common method of disposal of dairy parlour washings is by land spreading. This treatment method has numerous problems, namely high-labour requirements and the potential for eutrophication of surface and ground waters. Constructed wetlands are commonly used for treatment of secondary municipal wastewaters and they have been gaining popularity for treatment of agricultural wastewaters in Ireland. Intermittent sand filtration may offer an alternative to traditional treatment methods. As well as providing comparable treatment performance, they also have a smaller footprint, due to the substantially higher organic loading rates that may be applied to their surfaces. This paper discusses the performance and design criteria of constructed wetlands for the treatment of domestic and agricultural wastewater, and sand filters for the treatment of domestic wastewater. It also proposes sand filtration as an alternative treatment mechanism for agricultural wastewater and suggests design guidelines.     

Synchrotron radiation for direct analysis of metalloproteins on electrophoresis gels

Metalloproteomics requires analytical techniques able to assess and quantify the inorganic species in metalloproteins. The most widely used methods are hyphenated techniques, based on the coupling of a high resolution chromatographic method with a high sensitivity method for metal analysis in solution. An alternative approach is the use of methods for solid sample analysis, combining metalloprotein separation by gel electrophoresis and direct analysis of the gels. Direct methods are based on beam analysis, such as lasers, ion beams or synchrotron radiation beams. The aim of this review article is to present the main features of synchrotron radiation based methods and their applications for metalloprotein analysis directly on electrophoresis gels. Synchrotron radiation X-ray fluorescence has been successfully employed for sensitive metal identification, and X-ray absorption spectroscopy for metal local structure speciation in proteins. Synchrotron based methods will be compared to ion beam and mass spectrometry for direct analysis of metalloproteins in electrophoresis gels.     

Phosphate uptake and utilization by bacteria and algae

Bacterial uptake of inorganic phosphate (closely investigated in Escherichia coli) is maintained by two different uptake systems. One (Pst system) is P_i-repressible and used in situations of phosphorus deficiency. The other system (Pit system) is constitutive. The Pit system also takes part in the phosphate exchange process where orthophosphate is continuously exchanged between the cell and the surrounding medium.Algal uptake mechanisms are less known. The uptake capacity increases during starvation but no clearly defined transport systems have been described. Uptake capacity seems to be regulated by internal phosphorus pools, e.g., polyphosphates. In mixed algal and bacterial populations, bacteria generally seem to be more efficient in utilizing low phosphate concentrations. The second half of this paper discusses how bacteria and algae can share limiting amounts of phosphate provided that the bacteria have pronouncedly higher affinity for phosphate. Part of the solution to this problem may be that bacteria are energy-limited rather than phosphate-limited and dependent on algal organic exudates for their energy supply.The possible phosphate exchange mechanism so convincingly demonstrated in Escherichia coli is here suggested to play a key role for the flux of phosphorus between bacteria and algae. Such a mechanism can also be used to explain the rapid phosphate exchange between the particulate and the dissolved phase which always occurs in short-term ³²P-uptake experiments in lake waters.     

Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.     

Orthophosphate turnover in East African lakes

Summary Turnover rates of ³²P–PO₄ and concentrations of orthophosphate as soluble reactive phosphorus (SRP) were measured in five East African waters. Rapid incorporation of ³²P–PO₄ by the seston and orthophosphate concentrations below the limit of detectibility were found in Lakes Elmenteita, Naivasha, and Naivasha Crater Lake. Turnover was slow and orthophosphate concentration high in both Lake Nakuru and the Crescent Island Crater basin of Lake Naivasha. Further experiments in Lake Nakuru indicated that colloidal binding of orthophosphate was limited and that particles retained by an 8.0 filter incorporated 66% as much tracer as particles retained by a 0.1 filter. These experiments strengthen our conclusion that a large quantity of orthophosphate is available for algal use in Lake Nakuru.     

Rapid physiological assays for nutrient demand by the plankton. II. Phosphorus

Four bioassays for phosphorus demand were compared. ³²P-turnoverrates provided an integrated measure of transport capacity ofthe assemblage and ambient orthophosphate levels, but rateswere often not simply exponential and could not be accuratelyquantified by a single rate constant. ATP responses to phosphorusenrichment were transient and they poorly separated naturalplankton populations with very different degrees of P-deficiency.Alkaline phosphatase activity corresponded well with other measuresof phosphorus demand although low activities did not necessarilyimply non-limiting phosphorus conditions. Selective luxury accumulationof N and P by the seston from an N + P enrichment provided themost informative guide to the relative demand for the two elements.No single approach could be considered conclusive on its own,but in combination the assays provided a reliable measure ofphosphorus demand and deficiency within natural assemblagesof plankton.     

Prebiotic Phosphate Ester Syntheses in a Deep Eutectic Solvent

We report a route to synthesize a wide range of organophosphates of biological significance in a deep eutectic solvent (2:1 urea and choline chloride), utilizing various orthophosphate sources. Heating an organic alcohol in the solvent along with a soluble phosphorus source yields phosphorus esters of choline as well as that of the added organic in yields between 15 to 99 %. In addition, phosphite analogs of biological phosphates and peptides were also formed by the simple mixing of reagents and heating at 60–70 °C in the deep eutectic solvent. The presented dehydration reactions are relevant to prebiotic and green chemistry in alternative solvents.     

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [³H]glycerol, [³H]inositol, or [³²P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of `fingerprint' methanolysis of these phospholipids confirmed the identifications.