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1.
In cation exchange when the sample cations are weak electrolytes, several benefits arise from choosing the eluent cation to be a weak electrolyte with a similar pK. The entire pH buffering range of the eluent can then be practically utilized to effect a modulation of the sample elution volumes. For elution of aliphatic amino acids from sulfonated polystyrene resin using a pyrazolium chloride eluent (pH 4.0 to 1.5) we obtained selectivity coefficients that are independent of pH and ionic strength as well as only weakly dependent on temperature and sample concentration. As a result the sample elution behavior is accurately predictable. The analytical-scale data were used to develop a preparative procedure capable of resolving a sample to resin load 10 times larger than previously reported for amino acid chromatography on laboratory-scale systems. 相似文献
2.
Hans M. H. van Eijk Dennis R. Rooyakkers Bernadette A. C. van Acker Peter B. Soeters Nicolaas E. P. Deutz 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,731(2):730
Measurement of the incorporation of labeled amino acids in plasma albumin, isolated from plasma sampled at different time points after infusion start is a well-known technique to study human albumin synthesis. Unfortunately, no chromatographic method has been described yet, enabling the automated isolation of high-purity albumin from large numbers of plasma samples as is required to study the kinetics of this process. Therefore, we developed a fast protein liquid chromatographic method, capable of processing 200 μl amounts of plasma in 74 min (injection to injection). The system can run unattended as the FPLC system is connected to a sample processor equipped with a polyether ether ketone (PEEK) sample loop and a cooled sample tray. Albumin isolation was divided into three steps. First, plasma samples were injected onto a 1-ml Blue Sepharose HiTrap affinity column, equilibrated with 50 mmol/l phosphate buffer (pH 7.0). After elution of non-binding protein, switching the solvent to phosphate buffer with 1.5 mol/l sodium chloride eluted albumin. The resulting albumin fraction was desalted on-line by directing it through two consecutive HiTrap 5-ml desalting columns, whereafter it was retained in the system within a 5-ml PTFE loop, connected to a motor valve. After switching this valve, thus bypassing the sample loop, the phosphate buffers were changed automatically to Tris buffers. Final purification involved elution of the captured fraction over a 1-ml ion-exchange Resource Q column, using a sodium chloride gradient, ranging from 0 to 0.5 mol/l in Tris buffer (20 mmol/l, pH 7.5). A more than 99% purity of the final albumin fraction was confirmed by capillary electrophoresis. 相似文献
3.
Study on the purification of polysaccharides from Noscoc flagelliforme with radial flow chromatography 总被引:1,自引:0,他引:1
Yu-Jie Dai Jing-Wen Wang Shi-Ru Jia Si-Jun Yue Meng-Yao Jia Peng Xu 《Biotechnology and Bioprocess Engineering》2009,14(3):377-382
The isolation and purification of polysaccharide from Noscoc flagelliforme by radial flow chromatography were studied. The column (7.7 cm of bed length and 229.6 cm3 of bed volume) was packed with DEAE-01 anion ion-exchange resin and gradient eluted with NaCl solutions. The content of the
polysaccharide was determined with the phenol-sulfuric acid method. The effects of sampling weight, elution velocity, and
elution concentration gradient on the separation efficiency were examined and three isolated peaks were obtained. The optimal
separation conditions are 10 mg of the sampling weight (sampling volume is 20 mL), 1.0 mL/min of the elution velocity, and
1.00 mol/L2 of NaCl gradient elution. The adjacent peak resolutions among the three main components (1, 2, and 3 according to their elution
order) are 0.660 (R
12) and 0.786 (R
23), respectively. It is deduced that 39.8 cm of the bed length is required for the fully separation of the three polysaccharides.
An erratum to this article can be found at 相似文献
4.
The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation. 相似文献
5.
Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds 总被引:2,自引:0,他引:2
A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino, keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. 14C-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure. 相似文献
6.
The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM
plasma membrane
- TPP+
tetraphenylphosphonium ion
-
pH
pH gradient
-
membrane potential
This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations. 相似文献
7.
Masahiko Akiyama Akira Shimoyama Cyril Ponnamperuma 《Origins of life and evolution of the biosphere》1982,12(2):215-227
Amino acids were recovered at a concentration level of 10–9 M/g from the interior of chert and dolomite of the Late Precambrian Thule Group. Examination of the stability of amino acids in chert under dry-heating conditions suggests that these amino acids have been preserved with a predominance of L-enantiomers in the Precambrian chert. Enantiomer analysis of amino acids in dolomite showed a thermal effect resulting from a late Precambrian igneous intrusion. This evidence indicates that the amino acids isolated from the Thule samples were chemical fossils and not recent contaminants. 相似文献
8.
C. W. Hancher E. F. Phares G. David Novelli A. D. Kelmers 《Biotechnology and bioengineering》1969,11(6):1055-1070
An engineering-scale procedure for the recovery of 300–400 g batches of mixed transfer ribonucleic acids is described. Semicontinuous growth of E. coli K-12 MO7 yielded 77 kg of harvested cells in four days. Phenol extraction and ethanol precipitation recovered a crude tRNA material that was further purified by DKAE-cellulose chromatography in runs of 1 × 106 A260 units each on a 6 × 30 in. column using a 240 1, gradient elution. The purified tRNAs were partially concentrated and resolved into three groups. 相似文献
9.
System y+L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na+ replacement with K+ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y+L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various
amino acids in the presence of Na+, K+, Li+ and guanidinium ion. In agreement with the prediction, the specificity of system y+L was altered by the monovalent cations. In the presence of Na+, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K+, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li+ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na+; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards
β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was
restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids.
The results suggest that the monovalent cations stabilize different carrier conformations.
Received: 22 January 1996/Revised: 26 April 1996 相似文献
10.
Analysis, Production, and Isolation of an Extracellular Laccase from Polyporus anceps 总被引:4,自引:3,他引:1
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Richard J. Petroski Wanda Peczynska-Czoch John P. Rosazza 《Applied microbiology》1980,40(6):1003-1006
Methods are described for the analysis, production, and isolation of laccase produced by a strain of Polyporus anceps. A simple quantitative colorimetric assay based on the oxidation of syringaldazine to syringaldazine quinone is described. Using a defined medium supplemented with the amino acids cysteine and histidine and with elevated phosphate, consistently high titers of laccase were obtained. The enzyme was isolated directly from fermentation medium by binding to diethylaminoethyl cellulose, and, once bound to the ion exchanger, it could be stored for 6 months at -70°C with minimal loss of activity. The enzyme was quantitatively recovered from the resin by elution with 0.2 M phosphate buffer (pH 5.0). 相似文献
11.
The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates 总被引:3,自引:1,他引:2
Abstract— The effects of phenylalanine and other amino acids on incorporation of several different 14C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different 14C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [14C]tyrosine, and (2) leucine, which inhibited incorporation of [14C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant. 相似文献
12.
In this paper, adsorption behaviors of typical neutral (alanine), acidic (glutamic acid) and basic (lysine) amino acids onto the surfaces of neutral as well as positively and negatively charged silver chloride nanoparticles were examined. Silver chloride nanoparticles with different charges and different water content were synthesized by reverse micelle method. The adsorptions of the above mentioned amino acids onto the surfaces of differently charged silver chloride nanoparticles were found to depend strongly on various parameters including pH of the aqueous solution, type of amino acid, water to surfactant mole ratio, and type of charges on the surfaces of silver chloride nanoparticles. It was found that the interaction of –NH3 + groups of the amino acids with silver ion could be a driving force for adsorption of amino acids. Alanine and Glutamic acid showed almost similar trend for being adsorbed on the surface of silver chloride nanoparticles. Electrostatic interaction, hydrophobicity of both nanoparticle and amino acid, complex formation between amine group and silver ion, interaction between protonated amine and silver ion as well as the number of nanoparticles per unit volume of solution were considered for interpreting the observed results. 相似文献
13.
G. Raya-Tonetti P. Córdoba J. Bruno-bárcena F. Siñeriz N. Perotti 《Biotechnology Techniques》1999,13(3):201-205
A strong anionic exchange resin was used to recover lactic acid directly from fermentation in an upflow fluidized bed column, resulting in 0.18 g lactic acid/g resin bound with a subsequent elution of 94%. When the culture broth was heated and adjusted pH to 8.0, 0.4 g lactic acid was bound per g resin, with a subsequent elution of 90%. L(+) and D(–) lactic acid isomers distribution was analyzed in the elution product resulting in an increase of L(+) isomer concentration. The resin did not alter its binding capacity after 23 cycles. 相似文献
14.
Ajit J. Shah Verner de Biasi Steve G. Taylor Claire Roberts Panida Hemmati Richard Munton Andrew West Carol Routledge Patrick Camilleri 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,735(2):289
An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN−). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain. 相似文献
15.
以桑椹中黄酮类物质的吸附量和解吸率为指标,对比分析HZ-801、HZ-816、HZ-818等12种大孔吸附树脂对桑椹提取液的分离纯化效果,优选出最佳树脂HZ-801并通过对上样液pH、上样液质量浓度、上样量、吸附流速、洗脱剂质量浓度、洗脱剂用量、洗脱流速等影响因素的考察,确定最优工艺:吸附阶段上样液pH=4,上样液质量浓度0.45mg/mL,上样量420mL,吸附流速120mL/h,动态吸附量(干树脂)25.34mg/g,吸附率84.25%;洗脱阶段的洗脱剂体积分数为60%乙醇,洗脱剂用量270mL,洗脱流速120mL/h。此优化工艺条件下的洗脱率为85.78%,总黄酮纯度从23.64%提高到82.36%。 相似文献
16.
Summary Lactic acid produced by continuous culture of L.casei in an upflow packed bed reactor, was recovered with Amberlite IRA 400 in a fluidized bed column. Bed expansions of 1.25 and 2.25 were applied. Reutilization did not alter the capability of net recovery of 0.048 ± 0.01 g lactic acid/g resin. When 2200 cm/h of ascensional velocity was used, (bed expansion of 2.25), the resin adsorbed 39.3% of the initial lactic acid and 63.5% was eluted. This resin supported the highest exchange capacity of 0.126 g lactic acid/g resin. Applying high flow rates, the process has potential industrial applications due to the short time employed. 相似文献
17.
SYNOPSIS. The basic proteins of Paramecium aurelia nucleus were extracted from isolated nuclei and deoxyribonucleoprotein (DNP) of such nuclei. About 35–40% of the nuclear protein, predominantly a lysine-rich histone, is acid soluble. Five major components of the histone can be distinguished by polyacrylamide gel electrophoresis. Some components of Paramecium histone are similar to those of mammalian histones in their electrophoretic mobility, but they differ from the latter in the electrophoretic velocity and relative levels. The basic to acidic amino acid ratio of the histone from the ciliate is ~1.1–1.5, and its amino acid composition resembles closely that of yeast histone. Through the use of Sephadex G-200 gel filtration for purification of the histones extracted directly from isolated nuclei 2 basic proteins were resolved—component I, with an elution volume of 1.4, constitutes ~20% and component II, with an elution volume of 1.9, ~80%. 相似文献
18.
C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development
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Partitioning and translocation of 14C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of 14CO2 for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total 14C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered 14C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the 14C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The 14C-organic acids comprised from 3% to 14% of the soluble 14C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. 14C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while 14C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of 14C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that 14C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling. 相似文献
19.
Summary We explore here the special structural features of certain diamino acid analogs which may account for their intense accumulation into tumor cells, first observed for the Ehrlich ascites tumor cell for in vitro suspensions. This accumulation, which ordinarily occurs mainly by system A for its dipolar substrates, is so intense for these tripolar diamino acids accompanied by the chloride ion as well as by displacement, especially of the cellular potassium ion, that the cells swell to several times their normal volume and osmotic destruction arises. These structural features receive our reconsideration here toward understanding the energization of amino acid transport into cells, also toward identifying among them possible superior 11C-labeled tracers for imaging tumors in situ by positron emission tomography (PET). The possibility of therapeutic, perhaps osmotic, destruction of inoperable terminal gliomas by topical application of such amino acids by microdialysis has also been considered in preliminary tests by one of us (G.R.) and his associatesPresented at Uppsala Symposium on Positron Emission Tomography, 9/13/91 相似文献
20.
《Comparative biochemistry and physiology. A, Comparative physiology》1987,86(2):273-279
- 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
- 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na+-ions and Cl−-ions were required for the amino acid transport.
- 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
- 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
- 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.