Contribution of bacterial cell nitrogen to soil humic fractions

Summary Living cells ofSerratia marcescens, uniformly labelled with¹⁵N, were added to samples of maple (Acer saccharum) and black spruce (Picea mariana) forest soils. After different periods of incubation from zero time to 100 days, the soils were subjected to alkali-acid and phenol extraction to provide humic acid, fulvic acid, humin and humoprotein fractions. Significant amounts of the cell nitrogen were recovered in the humic and fulvic acids immediately after addition. After incubation, less cell, nitrogen appeared in the humic acid and more in the fulvic acid. The amount of cell nitrogen recovered in the humin fraction increased with incubation. Roughly 5 to 10 per cent of the added cell nitrogen was found as amino acid nitrogen from humoprotein in a phenol extract of the humic acid. The data are consistent with the occurrence of co-precipitation of biologically labile biomass nitrogen compounds with humic polymers during the alkaline extraction procedure involved in the humic-fulvic fractionation.     

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.     

Nitrogen cycling in grasslands at Kanpur,India

Summary The present paper deals with the distribution of nitrogen in the different plant compartments and in the top 30 cm soil among the protected, semi-protected and open-grazed grasslands at Kanpur (26° 26 N latitude and 80° 22 E longitude).The protected site indicated greater nitrogen content (g N m^–2) in the aboveground and belowground plant parts as compared to those of semi-protected and open-grazed sites. Nitrogen content in the combined live and dead herbage varied from 2.6 to 53.5 g m^–2 in protected community, 1.6 to 27.6 g m^–2 in semi-protected and 0.9 to 17.4 g m^–2 in open-grazed community. The content ranged between 1.0 to 17.7, 0.5 to 9.7 and 0.4 to 6.6 g m^–2 for belowground and from 0.1 to 1.1, 0.1 to 0.5 and 0.1 to 0.3 g m^–2 for litter compartments in protected, semi-protected and open-grazed community respectively.A significant positive relationship was found with the nitrogen per gram dry weight in combined live and dead herbage of the study sites and the average monthly relative humidity.The distribution pattern of nitrogen in plant/soil system indicated that the major portion of nitrogen (91 per cent in the protected, 95 per cent in the semi-protected and 96 per cent in the opengrazed stands) in the system was retained in the soil while a small fraction of it (9 per cent, 5 per cent and 4 per cent on protected, semi-protected and open-grazed area respectively) resided in plant compartments. Partitioning, uptake, transfer and release of nitrogen have been discussed in detail for all three sites.     

Reactions between amino acid compounds and phenols during oxidation

Summary About 30 per cent of organic soil nitrogen can be hydrolized with HCl to amino acids; about 30 per cent is nonhydrolizable. In contrast to this high content of amino acid nitrogen is the small availability of the nitrogen to micro-organisms. In light of the theory proposing a reaction between the -amino group of amino acids or peptides and quinones formed during oxidation of lignin degradation products or other phenolic compound, different types of phenols were oxidized by phenolases in presence of amino acid compounds.It could be shown that the reaction of binding of nitrogen started at pH values higher than 6.5, and that only such phenols reacted which had no methoxylated hydroxyl groups. The reaction of some phenols during oxidation in presence of amino acids was accompanied by deamination and decarboxylation of the latter.The reaction products of phenols with amino acids were stable against hydrolysis. Using peptides it was found that all amino acids, except the N-terminal which is bound to oxidized phenols, could be hydrolyzed normally.With serum albumin it could be shown that there is a reaction with the amino group of the N-terminal amino acid and also with the -amino group of lysine residues with phenols during oxidation. The reacted protein seemed to be degraded normally with a protease ofBacillus subtilis.Guest Scientist as Fulbright Research Scholar from the Agronomy Department of the Iowa State University, Ames, Iowa, U.S.A.     

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

The growth of wheat plants in humic acid solutions under axenic conditions

Summary A technique is described for growing wheat plants in nutrient solutions containing C¹⁴-labelled humic acid under axenic conditions. The general appearance of axenic plants was indistinguishable from plants grown in association with microbes. C¹⁴-labelled humic acid enhanced the growth of both roots and shoots showing that by-products of microbial degradation of humic acid are unnecessary for this enhanced plant growth. Thus humic acid had a direct effect on the growth processes. The C¹⁴-labelled humic acid was taken up by the roots and virtually none was transported to the shoot. Only some 30 to 40 per cent of the incorporated radioactivity was associated with the root cell walls and thus more than 60 per cent was in the cytoplasm and may have influenced the biochemical processes involved in the regulation of plant growth.     

Preparation,purification, and properties of E. coli virus T2

1. A method for the preparation of 8 to 10 liter quantities of T₂ virus lysates, titering 2 to 5 x 10¹¹ infectious units per ml. has been described. 2. Procedures have been developed for the concentration and purification of virus to a high specific infectivity. No fractionation procedure of the several used succeeded in further raising the specific infectivity of these purified preparations. 3. Some of the general properties of the better preparations have been determined. They exhibited titers of 2 x 10¹⁵ infective units per gm. of material or 1.2 x 10¹⁶ per gm. of nitrogen. 4. A study of the distribution of nitrogen among the various fractions of the virus showed that about 6 per cent of the total nitrogen is soluble in 4 per cent trichloracetic acid; that the protein nitrogen is about 40 per cent of the total and the nucleic acid nitrogen is 53 per cent. At least 96 per cent of the total phosphorus is in the nucleic acid fraction. Less than 0.5 per cent quantities of lipid and PNA were found.     

Nitrogenous Compounds and Nitrogen Metabolism in the Liliaceae: III. Changes in the Soluble Nitrogen Compounds of the Tulip and their Relation to Flower Formation in the Bulb

The principal free amino-acid of the vegetative tulip bulb isarginine and the alcohol insoluble nitrogen fraction containscombined arginine to the extent of 47 per cent. of the nitrogen.In the green leaf the amides glutamine and asparagine occurwhereas free arginine is virtually absent. These amides do notoccur appreciably in the vegetative bulb. By appropriate temperature treatments flowers were initiatedand after the floral parts were formed they were caused to developto the point of emergence from the bulb. The concomitant changesin the soluble nitrogen compounds were determined. The dramatic change in the composition of the soluble nitrogeninvolving a shift from the predominance of arginine to a relativeincrease in glutamine and asparagine occurs when the alreadyinitiated flowers develop. -methyleneglutamine and -methyleneglutamic acid which are prominentin the tulip seem not to be greatly affected by the treatmentsthat cause floral initiation and development. Their metabolismseems superimposed upon the nitrogen metabolism based on argininein the bulb and amides in the leaf which is responsive to thetreatments involved in flower formation.     

Contribution to the estimation of nucleic acids in wheat roots

Several methods of NA isolation and estimation were examined in order to determine RNA in the whole root systems of young wheat plants. It was found out that during the hydrolysis of roots with perchloric acid according toOgur andRosen (1950),Spirin (1958) andHeitefuss (1965) a considerable amount of orcine-positive compounds is released which cannot be adequate to the RNA content. Therefore the separate RNA determination in the presence of DNA was excluded even after the NA fractionation by hydrolysis at various temperature and perchloric acid concentration. Besides NA hydrolysates contained a high amount of other compounds absorbing in the UV-region. Compounds interfering with both these methods were present especially in the basal parts of roots. Using the method ofKern (1960) a nucleoprotein fraction was isolated containing about 75 per cent of the total NA content in the tissue. This fraction consisted of more than 90 per cent RNA combined with proteins, namely the ribosomal RNA. After the hydrolysis it could be estimated spectrophotometrically for the hydrolysate did not contain other compounds absorbing in the UV-region. In the root tips the content of this fraction was high in comparison with the basal parts of roots and it changed with the kind of nutrition.     

Concentration of acid phosphatase,ribonuclease, desoxyribonuclease,beta-glucuronidase,and cathepsin in droplets isolated from the kidney cells of normal rats

1. Three fractions of "droplets" having diameters of 1 to 5 micro (fraction I), 0.5 to 1.5 micro (fraction II), and 0.1 to 1.0 micro (fraction III) were isolated from the kidney cells of normal rats. 2. All three "droplet" fractions showed 10 to 15 times higher activities of acid phosphatase, beta-glucuronidase, ribonuclease, desoxyribonuclease, and cathepsin than the total homogenate and the mitochondrial fraction. 3. After a rough fractionation of the total homogenate, approximately 50 per cent of the 5 enzymes was found in the fractions which contained the "droplets" and approximately 30 per cent in the supernatant fluid. 4. The similarities between the enzymatic properties of the "droplets" from kidney cells and of the fractions isolated from liver cells by other investigators have been discussed.     

Mineralization of nitrogen in two soil-organic-matter fractions extracted with formic acid

Summary Extraction of a calcareous meadow soil with boiling formic acid containing 10 per cent acetylacetone yielded a fraction of soil organic matter representing about 40 per cent of the total. This fraction contained only 2.2 per cent N. Less than 18 per cent of this was mineralized during incubation for 8 weeks at 28°C. Further extraction of the soil with a formic acid and hydrofluoric acid mixture yielded a second fraction. This represented about 37 per cent of the total organic matter and contained 6.0 per cent N of which up to 41 per cent was mineralized during incubation. Preincubation and leaching had a stabilizing effect on subsequent mineralization of native N of a standard sandy loam soil.     

THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U-14C]GLUTAMATE

Abstract—

• 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-¹⁴C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions.

• 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered.

• 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent.

• 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate.

• 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour.

• 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

     

Effectiveness of selection under different mating systems for the improvement of protein content in wheat (Triticum aestivum L. em Thell.)

Summary The effectiveness of selection for the improvement of protein content under random intermating (recurrent selection) and selfing series (pedigree selection) was evaluated in a cross of winter and spring wheats, Atlas 66 × HD 1977. Selection of 10 per cent high protein families resulted in an increase of 3.25 per cent and 4.30 per cent of the mean of checks through pedigree and recurrent selection, respectively. The mean protein differences in both methods were not significant. The increase in protein content was accompanied by a decrease in the grains per spike, grain yield and 1000-kernel weight, and the decline was relatively higher in recurrently selected than pedigreed population. Since the pedigree method is simple, less time consuming, economically cheaper, has favourable shifts in association and better correlated responses, it was decided to follow a few cycles of pedigree selection in early segregating generations, after which one or two cycles of recurrent selection in the elite lines could be introduced to increase genetic variation and concentrate favourable genes for grain yield.From a dissertation submitted by the senior author in partial fulfilment of the requirements for the Ph.D. degree. Work supported by financial assistance in the form of a Senior Research Fellowship received by the senior author from the I.C.A.R., New Delhi.     

Differential extraction of soluble pools from the cytosol and the vacuoles of yeast (Candida utilis) using DEAE-dextran

The plasma membrane of Candida utilis cells was rapidly disrupted by a small dose of DEAE-dextran. The vacuolar membranes, in contrast, remained intact under isotonic conditions. Therefore, the cytosolic pool could be extracted in a first step, and in a second step, after disruption of the vacuoles, the vacuolar pool. The two extracts were studied in cells grown on different nitrogen sources, namely ammonium, arginine, ornithine, citrulline, glycine, and proline.The amount of soluble amino acids in Candida cells varies considerably depending on the nitrogen source. This is largely caused by the variation in size of the vacuolar pool (0.8–2.4 mmol per g protein) containing nearly all nitrogen-rich amino acids such as arginine and ornithine, whereas the size of the cytoplasmic pool, holding most of the glutamic acid, is fairly constant (1.3 mmol per g protein). Upon nitrogen starvation the vacuolar pool was reduced much more than the cytosolic pool. A storage and buffer function of the vacuolar pool was also indicated by the much slower turnover of the vacuolar than of the cytosolic glutamine in an isotope labelling experiment. Potassium, sodium, orthophosphate, ATP, and other substances absorbing at 260 nm were found predominantly in the cytosolic extracts. Extraction of uniformly ¹⁴C-labelled cells showed that the total soluble pool of the cells contained about 10% of the total carbon. Of this about 45% was in the vacuolar the rest in the cytosolic extract. The labelled extracts were further characterized by ion exchange chromatography.Non-Standard Abbreviations DEAE-dextran diethylaminoethyl-dextran - MES 2-(N-morpholino)ethane sulfonic acid - PIPES piperazine-N,N-bis-2-ethane sulfonic acid - c-extract cytosolic extract - v-extract vacuolar extract     

Symbiotic nitrogen fixation and vegetative growth of cowpea (Vigna unguiculata (L.) walp.) in waterlogged conditions

Summary Various periods of waterlogging (up to 32 days duration) were imposed upon cowpea plants grown in pots under controlled glasshouse conditions. Particular attention was paid to treatment effects on nodule cortication, nitrogenase activity and fixation efficiency, and the consequent differences in plant dry weight and nitrogen content.All waterlogging treatments increased nodule cortication as compared with the unstressed controls; a 16-day stress period being of critical duration with respect to the bi-phasic nature of this anatomical response. Conspicuous lenticel-type protuberances were present on nodules formed under waterlogged conditions but were markedly reduced, or indeed absent, in the controls. Total dry weight of nodules per plant was reduced by 60 per cent after only 8 days waterlogging, but nitrogen fixation efficiency of nodules which persisted was only 18 per cent less than those on control plants; mean nodule cortex having increased from 39.8 (control) to 51.5 per cent. After 16 days waterlogging, total plant dry weight was decreased by ca 60 per cent as compared with control plants; reflecting similar adverse changes in leaf, stem and root dry weight. The most severe treatment (32 days waterlogging) did not further reduce plant dry weight but mean nodule cortex area increased from 55.9 (16 days) to a maximum of 59.3 per cent. With the exception of nodules, percentage nitrogen content of various plant components was unaffected by the treatments imposed.Both the formation of enlarged lenticles and increased nodule cortication are regarded as adaptive anatomical responses which facilitate continued symbiotic nitrogen fixation and vegetative growth of this legume under waterlogged conditions.One of a series of papers describing work undertaken in a collaborative project with the International Institute of Tropical Agriculture, Nigeria, sponsored by the U.K. Ministry for Overseas Development.     

Nitrogen Metabolism of Detached Kalanchoe Leaves in the Dark in Relation to Acidification, Deacidification, and Oxygen Uptake

Detached Kalancho leaves were placed in the dark and changesin the amounts of total soluble N, asparagine, glutamine, aminoN, ammonia, and titratable acid were followed during the periodsof acidification and deacidification. Abundant starch was presentthroughout the experiments. The amount of acid which accumulatedin the initial period was increased by placing the leaves in5 per cent. CO₂ and also by decreasing the temperature from25 to 10°. In neither case were the nitrogen fractions affectedin a similar way to the acid levels. No consistent relationshipcould be found between the amount or change in amount of acidand the amounts of the nitrogen fractions. Over prolonged periodsthe drifts in the amounts of the nitrogen fractions were verysimilar to those known in non-succulent leaves. It is concludedthat, despite the large amounts of free acid and starch present,the nitrogen metabolism of detached Kalancho leaves in the darkis similar to that of non-succulent leaves. A possible explanationis suggested for the incorporation of ¹⁴C from ¹⁴CO₂ into nitrogenoussubstances in Kalancho leaves during dark acidification. Changesin the amounts of glutamine, which seemed to be related to oxygenuptake rather than to acid fluctuations, were measured in comparableleaf samples showing different rates of oxygen uptake. The rateof oxygen uptake of leaves and leaf disks in the dark was controlledby placing them in an atmosphere of nitrogen, or 5, 20, or 100per cent, oxygen, by the addition of 10^–3 M. cyanide,or by varying the temperature. At the end of a given periodthe amount of glutamine was always greater in the samples whichhad shown the higher rate of oxygen uptake during this perod.This correlation was found irrespective of whether the amountof glutamine had increased or decreased. With the assumptionsthat glutamine production and consumption were proceeding simultaneouslyand that the rate of consumption was unaffected by the rateof oxygen uptake, the experimental values are consistent withthe hypothesis that the rate of glutamine production and therate of oxygen uptake were directly related. The possible significanceof these findings is discussed.     

The growth of fractal humic acids: Cluster correlation and gel formation

The growth of humic acids, prepared by a gentle method from two different kinds of soils (I and II), has been studied using small-angle neutron scattering at an acidity corresponding to pH 5.0 and 0.10 M ionic strength (NaCl). Humic acids aggregate either to large clusters with a fractal dimension of 2.3₅ and an average diameter of 1720 (I) or to clusters with an average diameter of 700 (II). After storage for 2 days at 4 °C, the latter aggregates (II) formed a gel. In a step toward gelation, we observed cluster-cluster interaction from the neutron-scattering data in the form of a correlation peak. These differences in size can be explained by assuming that the smaller particles (II) are trapped into a nonequilibrium state characterized by the temperature-solvent condition. The importance of a humic acid gel network for the conservation of water and nutrients in the environment is discussed.     

The Labelling of Malic and Citric Acids of Pea Leaves and Cotyledons of Germinating Peas by 14CO2 in the Dark

Leaflets of green pea plants and cotyledons of germinating peaswere kept in the dark in air containing ¹⁴CO₂. The malate extractedwas labelled to 35–40 per cent in the C-1 position andthe citric acid was labelled to 70–75 per cent in theC-6 position. This showed that two carboxylations, at least,were involved in both tissues, i.e. probably of a three-carbonacid to form malate, and of -oxoglutarate to form isocitrate.     

Chemical forms of plant and soil iron as influenced by soil moisture

Summary Experiments were conducted to determine whether soil moisture content has an effect on the chemical forms of plant and soil iron. Soybean plants, variety Lee, were grown on Adelanto loam soil under greenhouse conditions. Two different moisture levels, 75 per cent and 120 per cent of the moisture equivalent, were maintained in soil samples placed in individual containers. The same moisture treatments were used for separate soil samples on which the test plants were grown.Soil iron forms were determined in the moisture-treated soil by using different extracting agents. A significant decrease in soil iron extracted with 10^–4 M EDTA from soil at the high moisture level was attributed to a relative increase in the free calcium ion.Soybean plants grown under the high moisture level were chlorotic while those under the low moisture level were green in appearance. Plant samples were taken at two stages of growth for subsequent analysis.The chemical analysis of the leaf tissues have shown the presence of equal amounts of total iron and less amounts of water-soluble and active iron in chlorotic tissues as compared to non-chlorotic tissues. The difference found between chlorotic and non-chlorotic plants in the amount of iron in the water extract was in the trichloroacetic acid-soluble fraction. The water- and salt-soluble protein nitrogen was approximately the same in chlorotic and non-chlorotic leaves.     

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 µm diameter fraction believed to be composed largely of basal cells, and a 15 µm diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 µm cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 ± 2.7% ciliated cells with 6.5 ± 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 ± 2.1%) and Clara cells (27.0 ± 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 ± 0.09 × 10⁶ cells/ trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 µm cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 ± 66 pmol/ min/ mg protein or 51.2 ± 20.5 pmol/ min/ 10⁶ cells for 7-ethoxycoumarin deethylase and 31.7 ± 15.4 pmol/ min/ mg protein or 10.5 ± 4.8 pmol/ min/ 10⁶ cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.Abbreviations CD cell digest - DNase deoxyribonuclease I - E-1 first elutriator fraction - E-2 second elutriator fraction - E-3 third elutriator fraction - 7-Ec 7-ethoxycoumarin - FCS fetal calf serum - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - HpBS HEPES-buffered salt solution - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PEG Carbowax polyethylene glycol 6000