The determination of α-amino acids in seawater using a fluorimetric analyser

A method is described for the detection of free α-amino acids in the picomole range in seawater samples of less than 100 ml volume. The modifications made to a standard amino acid analyser to incorporate a fluorimetric detection system are described in detail, together with the methods for desalting the seawater samples on cation exchange resin and the concentration procedures prior to analysis. A complete analysis of up to 30 amino acids requires around 3 h with a detection limit of around 0.05 μg of an individual acid per litre. Twenty samples of seawater from different depths in the open Baltic have been analysed for their FAA contents together with 3 samples taken from the Kiel Fjord. The total FAA content of the samples ranges from 4.5 to 84 μg/l with a mean of around 25 μg/l. Five samples were hydrolysed prior to analysis in order to estimate the CAA content of the seawater. The values lay in the range 438–805 μg/l.     

A MICRO-FLUORESCENT METHOD FOR THE DE TERMINATION OF FREE AMINO ACIDS IN SEA-WATER AND THE DISTRIBUTION OF TOTAL FREE AMINO ACIDS

O-phthaldialdehyde is used for the determination of free amino acid in seawater by measuring the fluorescence produced by its reaction with the a-amino group of amino acids. This method is so sensitive that as little as 1-10 nM of amino acids and less than 1 ml of seawater may be sufficient for one test.This method is simple, rapid and accurate and a-amino acids contained in seawater can directly be determined without concentration and extraction. It is free from the interference of large content of inorganic salts and other nitrogenous compounds in seawater.A hundred and fifty-two samples collected at 24 stations in offshore of Zhejiang have been analyzed. The results obtained show that the range of concentration of the total free amino acid is 0.37-4.70 μM. The values are high near shore and low offshore.     

Sensitive fluorometric procedure to determine individual amino acids in marine waters

Improved methodology for amino acid analysis coupled with semi-micro isolation techniques make feasible the measurement of individual amino acids from marine waters where sample volumes are limited (e.g., sediment pore water or culture filtrates). If a water sample contains more than 20 nmole 1^?1 of the respective compounds, dissolved free amino acids (DFAA) can be isolated from 5 ml of filtrate by cation exchange and measured on a sensitive amino-acid analyzer equipped with a fluorometric detector. Two ml generally provides sufficient sample to determine total dissolved amino acids. Lower concentrations of amino acids can be measured after concentration from larger samples (250 ml) by ligand exchange.     

Marine proteomics: generation of sequence tags for dissolved proteins in seawater using tandem mass spectrometry

Dissolved proteins in seawater samples from the Gulf of Mexico were concentrated using tangential flow ultrafiltration and methanol/chloroform/water precipitation. Following concentration and purification, two different separation methods were employed. In one method, intact proteins were separated by SDS–PAGE and digested enzymatically in-gel. In the second method, the peptides resulting from a solution proteolytic digest of the whole protein pellet mixture were separated by capillary HPLC. In both methods, the final chromatographic separation was coupled on-line with a mass spectrometer using an electrospray interface, and peptide CID spectra were collected using tandem mass spectrometry (MS). De novo sequencing of the peptide tandem mass spectra generated short amino acid sequences (peptide tags) that were used to search databases for protein class and source information. Trends of conserved sequences for two specific classes of proteins were observed: membrane/envelope proteins and enzymes. Similarity searching of peptide tags produced identification of conserved sequences from several protein homologues originating from many different species, including: long chain fatty acyl CoA synthetase, anthranilate synthase, ribulose bisphosphate carboxylase, and luminal binding protein. These results provide new insight into the sources and production mechanisms for dissolved organic matter (DOM), as there is direct evidence for dissolved proteins other than the bacterial outer membrane proteins reported by Tanoue et al. Furthermore, the data presented herein support the idea that physical protection and selective preservation are not mutually exclusive survival mechanisms, but rather these two models are dependent upon one another for explaining the survival of refractory dissolved proteins in seawater.     

Metal-organic complexes in seawater pumped up from under the ground

Metal-organic complexes of transition elements removed by Amberlite XAD-2 resin from seawater pumped up from under the ground were determined. The proportions of iron, copper and zinc retained on the resin to dissolved forms of these metals were about 70, 40 and 5%, respectively, while manganese, cobalt and nickel were not retained on the resin. These results suggest that although iron may be retained in colloidal form, a significant fraction of copper is present in some organic form(s).     

Determination of copper complexation with natural organic ligands in seawater by equilibration with MnO2 II. Experimental procedures and application to surface seawater

The MnO₂ adsorption method combined with voltammetry is proposed for the direct determination of metal complexation in seawater of various salinities as a more satisfactory alternative to direct voltammetric measurements and bioassay methods. A small quantity of MnO₂ is equilibrated with copper ions in filtered seawater. Natural organic ligands in the seawater compete for copper with the MnO₂. Total dissolved copper is measured by differential pulse anodic stripping voltammetry after filtration and acidification of the sample. Preconcentration of natural water samples is unnecessary and measurement is performed at the natural equilibrium pH of the aerated sample. The analytical limit of detection of the method depends on contamination from the filtration step, and for copper complexation a ligand concentration of 5 × 10^?8 M was obtained. The sensitivity can be increased by use of radioisotopes as tracers. The method is very versatile in that complexation of various metals may be determined by any analytical method that measures total dissolved metal concentrations. Neither organic ligands nor their complexes with copper adsorb on the MnO₂ at pH8, but at pH 1.8 MnO₂ is an efficient scavenger for electroactive organic material.Samples of surface water from the Irish Sea and the Atlantic Ocean were found to contain ligand concentrations of 1.7 × 10^?7 and 1.1 × 10^?7 M, with conditional stability constants (log values) of 9.84 ± 0.13 and 9.86 ± 0.23, respectively, at pH 8.0.     

HPLC柱前衍生法测定海水中溶解态游离氨基酸

本文采用反相高效液相色谱法测定海水中溶解态游离氨基酸,整个分析过程只需40 min就可使所有海水样品中的氨基酸得到很好分离,对各含6pmol的16种氨基酸混合样分析偏差在10%左右,检测极限为100fmo1,线性范围在1pmol到Inmol之间。     

Measurement of manganese, zinc and cadmium complexation in seawater using Chelex ion exchange equilibria

Equilibria between Chelex 100* and manganese, zinc and cadmium ions were used to determine the complexation of these trace metals in 36‰ Gulf Stream seawater at 25°C and pH 8.2. The method utilized radiotracers (⁵⁴Mn, ⁶⁵Zn, and ¹⁰⁹Cd) to quantify trace metal adsorption from trace metal-amended seawater and from seawater containing a series of ethylenediaminetetracetate (EDTA)—metal ion buffers. Results were consistent with Chelex adsorption of both trace metal ions and trace metal—EDTA chelates. Equilibrium models fitted to the data were used to establish conditional stability constants for Chelex adsorption of manganese, zinc and cadmium ions and for adsorption of EDTA-chelates. These models also yielded ratios of free metal ions to total dissolved trace metal concentrations in seawater: 10^−0.1 for manganese, 10^−0.2 for zinc, and 10^−1.5 for cadmium. Independent measurements with a cadmium ion-selective electrode also yielded a free: total cadmium ratio of 10^−1.5.     

Dissolved free amino acids in coastal seawater using a modified fluorometric method

The method of Parsonset al. (1984) for measuring dissolved free amino acids (DFAA) in coastal seawater was modified. We found considerable interference in DFAA determination from ammonia dissolved in coastal seawater, although the interference of urea could be ignored. For DFAA analysis for coastal seawater samples, ammonia determinations for the same sample are needed to correct DFAA values. For coastal surface seawater samples from all over the Seto Inland Sea, Japan, values of DFAA ranged from undetectable to 1.87 μg-at N/l when corrected for ammonia, while uncorrected values ranged from undetectable to 2.61 μg-at N/l. DFAA, urea, nitrate+nitrite, ammonia and DON concentrations in surface seawater collected in the Seto Inland Sea were analyzed simultaneously. DFAA at four seasons constituted from 1.4 to 10.1% of DON, with a mean value of 6.5%. The concentration of urea was similar to that of DFAA and often higher than that of ammonium, although generally lower than that of nitrate.     

Variations of particulate proteins and dissolved amino acids in coastal seawater

The relationships existing between the protein-containing fraction of particulate matter and amino acids dissolved in seawater were studied in the Gulf of Marseille at different periods of the year. The concentration of particulate proteins was almost zero in February and attained maximum values during April and May, the average concentrations of dissolved amino acids (total) varied between 900 and 1200 nmole l^?1 but larger variations were encountered at the surface and in the vicinity of the sediment. The influence of meteorological conditions and the effect of the sediment on the distribution of nitrogenous substances were taken into account. Combined dissolved amino acids were more abundant than free dissolved amino acids in 90% of the cases. The concentrations of dissolved amino acids observed in a zone bordering the North Mediterranean are comparable to those found in other regions of the world.     

秋季南黄海表层海水中溶解氨基酸的分布与组成研究

用高效液相色谱法对2010年9月南黄海33个站位表层海水中总水解氨基酸(THAA)、溶解游离氨基酸(DFAA)、溶解结合氨基酸(DCAA)的分布、组成以及它们与环境因子的相关性进行了研究。结果表明:THAA平均浓度为2.08μmol/L,DFAA平均浓度为0.39μmol/L,DCAA平均浓度为1.69μmol/L。THAA浓度的分布大致呈近岸高、远岸低的特点;DFAA分布的规律性较差,在东部海域(中心约在35°N,123°E)出现高值。相关性分析显示溶解氨基酸与叶绿素a(Chl-a)、溶解无机氮(DIN)、细菌丰度、温度和盐度之间均无显著相关性。THAA中含量较高的个体氨基酸为丝氨酸、甘氨酸、天门冬氨酸、谷氨酸和丙氨酸,总和占70.59%。DFAA、DCAA中的主要成分与THAA基本相同。THAA中个体氨基酸的组成以34°N为界分为南北2个海区呈现明显的区域性特点。     

浙江西沪港重金属铜的配位容量和形态分析

根据2000年6月10～14日在浙江省西沪港采集的海水样品,利用AA-800石墨炉原子吸收分光光度技术和阳极溶出伏安法测定样品中重金属铜的含量,获得铜在海水中受不同的有机配体控制.不同粒级的铜表观配位容量表明西沪港海水过孔径1.00μm微孔膜的(ACuCC)较高,为144.4nmol/dm3;过0.40和0.20μm滤膜的(ACuCC)分别为103.0和102nmol/dm3;铜的有机配体条件稳定常数的对数值在7.25～9.14之间.铜的总量为21.72nmol/dm3.铜全部为稳定溶解态,其中pH2酸溶态占95.0%,强有机结合态占5.0%.溶解态铜中有机结合态占过滤海水中总铜的61.6%.     

小角刺藻生长过程中溶解游离氨基酸含量在海水中的变化

于1986年12月-1987年2月在温度为25±1℃下培养小角刺藻，以HPLC法测定该藻在培养过程中海水的溶解游离氨基酸（DFAA）含量变化。结果表明，小角刺藻不仅是海水中DFAA的主要生产者，而且也是DFAA的消耗者。小角刺藻不仅在缺乏硝酸盐的时候要吸收DFAA，即使在硝酸盐充足的情况下也会吸收。在小角刺藻的生长初期，水体中DFAA的含量迅速降低，而在后期却又大大增高。在小角刺藻的不同生长阶段，水体中DFAA的组成也不同。这些都可以说明海洋中DFAA的含量、组成和地区分布是密切地同浮游植物群落的繁殖消亡过程相关联的。     

Characterization of the proteinaceous matter in marine aerosols

Marine aerosols play a dominant role in the transfer of oceanic material to the atmosphere. Most marine aerosol originates when air bubbles burst at the sea surface ejecting material from the sea surface microlayer and bubble surface layers into the air. Concentrations of chemical compounds in these surface layers often differ from their concentrations in bulk water. We examined the enrichment of aerosols with proteinaceous matter and attempted to characterize the physical nature and sources of this matter. We measured concentrations of dissolved free (DFAA), dissolved combined (DCAA), and particulate (PAA) amino acids, transparent stainable particles (TSP), and bacteria and virus-like particles as carriers of protein, in natural and simulated aerosols. We also evaluated D/L ratios certain amino acids in all amino acid fractions.DFAA and DCAA enriched the aerosols we sampled by 1.2–20 times compared to bulk seawater; PAA enrichment was usually higher (up to 50-fold). Aerosols contained particles typical of seawater, e.g., microorganisms, organic debris, inorganic particles with adsorbed organic matter, but also a large number of semitransparent gel-like particles, which all contained amino acids. Some of these particles were probably scavenged from bulk water, but new particles produced as bubbles burst at the surface comprised at least 10% of total proteinaceous matter in the aerosol. D/L ratios of certain amino acid suggested that the particles were most likely made from dissolved polymers secreted by phytoplankton that were concentrated on bubble surfaces and in the microlayer. Examination with Alcian Blue (a dye that targets carbohydrates) and Coomassie Blue (a dye that targets proteins) showed that most TSP in the aerosols contained both proteins and polysaccharides. Microorganisms enriched the aerosols by up to two orders of magnitude, but contributed less than 4% to the total protein pool.     

Development of lipids during a spring plankton bloom in the northern North Sea: II. Dissolved lipids and fatty acids

During the spring plankton bloom (1976) in the northern North Sea the fatty acid and lipid distribution of the chloroform extractable fraction of seawater was analyzed by gas chromatography and thin-layer chromatography. The fatty acids in the seawater made up about 3% of the total dissolved organic matter. Maximum concentrations during the first phytoplankton bloom were in the range of 5 μmol C dm^?3. Palmitic acid (30.4%) and oleic acid (21.4%) as well as myristic, stearic and palmitoleic acids were the main fatty acids. The concentrations of fatty acids were higher below the thermocline in the deeper layers of the water column. The oleic acid showed large fluctuations, especially below the thermocline and seemed to be bound in a polar fraction. Different lipid classes containing fatty acids were determined by thin-layer chromatography. An estimation showed that the free fatty acid fraction made up the main portion (47.9%), followed by the triacylglycerol fraction (22.8%) and a more polar fraction with 25.1%. The triacylglycerol fraction appears to be more stable than the other fraction in the water column.     

A field comparison of two methods for the determination of copper complexation: Bacterial bioassay and fixed-potential amperometry

Complexation of copper added to seawater was determined by bacterial bioassay and fixedpotential amperometry. Consistent results were obtained by these two fundamentally different methods. The results of this study support the validity of both techniques and the field applicability of fixed-potential amperometry.The intercomparison studies were performed on samples collected at the N.Y.C. sewage sludge dumpsite and in relatively unpolluted coastal waters. In this limited study, the calculated free cupric ion concentrations at ambient total copper concentrations were similar at both sites.     

西北太平洋低纬度区域海水中溶解氨基酸的分布及组成研究

本文对2018年秋季西北太平洋低纬度区域上层海洋(5~200 m)中溶解氨基酸(THAA)的分布和组成进行了研究。结果表明,该海域表层海水中THAA的浓度范围为0.40~0.97μmol/L,平均浓度为0.58±0.14μmol/L;5~200m垂直断面上THAA的平均浓度为0.59±0.16μmol/L,范围为0.30~1.05μmol/L。调查海域内THAA浓度明显低于中国近海,在5~200m内的垂直分布基本表现出随深度增加而增加的趋势。将表层和垂直水体中的THAA分别与DOC、Chla等环境因子进行相关性分析,结果显示均无显著相关性。西北太平洋低纬度区域海水中的优势氨基酸是天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、甘氨酸(Gly)、苏氨酸(Thr)和丙氨酸(Ala)。基于氨基酸的碳归一化产率(THAA-C%)、降解因子(DI)值,表明该海域表层海水中的有机质降解程度较高,且随深度的增加而降低。     

Gas-liquid chromatographic determination of amino acids and vertical distribution of proteinaceous substances in sea water

Gas-liquid chromatographic method byGehrke et al. was applied to the determination of the dissolved proteinaceous substace. Sea-water samples were evaporated in vacuum to the volume of one twentieth of the initial volume and separated from deposited inorganic salt crystals. Thirteen kinds of amino acids except histidine, arginine and cystine were quantitatively determined for artificial sea water containing sixteen standard amino acids with accuracy of about five percent and with yield of 80 to 103 percent. This method has advantage comparing with paper or thin layer chromatography on the precision and comparing with ion-exchange method on sensitivity.The difference of amino acid concentration between the sea-water samples of different depth and different region was observed. The total amounts of dissolved amino acids were found to be between 33 and 92g per liter in sea water of Pacific Ocean taken at the surface to 1,500 m in depth.Contribution No. 41, from The Research Institute of North Pacific Fisheries, Hokkaido University.     

An improved method for the hydrolysis and MBTH analysis of dissolved and particulate carbohydrates in seawater

An improved method for the bulk characterization of particulate and dissolved combined carbohydrates in seawater is presented. Seawater samples are dried under vacuum and the residue is hydrolyzed with 12 M H₂SO₄. Carbohydrates in the hydrolysate are subsequently quantified spectrophotometrically by MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) analysis. Sulfuric acid was more effective in hydrolyzing high-molecular-weight structural carbohydrates (cellulose, chitin, and alginic acid) than weak (0.09 N) hydrochloric acid. Sulfuric acid hydrolysis of dissolved and particulate samples yielded estimates of carbohydrates equal to or greater (two- to four-fold) than those resulting from dilute (0.09 N) HCl hydrolysis. In samples from the Gulf of Mexico, total dissolved carbohydrates determined after sulfuric acid hydrolysis varied from 20 to 56 μM C and from 10 to 28% of the dissolved organic carbon determined by high-temperature catalytic oxidation.     

Spatial and temporal variations in free polyamine distributions in Uranouchi Inlet, Japan

Polyamines are an essential component of living organisms that influence normal cell growth and development. Despite the possible importance of polyamines for marine microbial ecology, concentrations of polyamines in seawater are not well known.Using HPLC analysis with a cation exchange column and OPA reagent, we determined polyamine concentrations in seawater without carrying out desalting, concentration or derivatization steps prior to chromatography.In seawater collected from Uranouchi Inlet, putrescine and spermidine were the predominant polyamines, ranging from undetectable (N.D.) to 4.4 nM and N.D. to 2.8 nM, respectively. Though other polyamines were detected, the frequency of detection was much lower than that of putrescine and spermidine; concentrations ranged from N.D. to 0.9 nM for cadaverine, and N.D. to 3.2 nM for norspermidine. Norspermine and spermine were detected in only a few samples; their concentrations varied from N.D. to 3.2 nM and N.D. to 0.5 nM, respectively. These concentrations were higher in the summer.