微波高压罐水解法在氨基酸分析中的应用简报

本文报导了采用微波高压罐水解技术快速水解食物蛋白质,并测定其１７种氨基酸含量的方法。研制的高压罐具有密闭性、微波穿透性好,耐热、耐压、耐腐蚀等特点。采用该高压罐研究了微波强度及作用时间等条件对氨基酸稳定性的影响,在微波输出功率１００Ｗ３５～４５分钟水解了３份食品样品,其氨基酸含量测定结果与传统的１１０℃２２左水解基本相符,１７种氨基酸５次平行测定结果ＣＶ值均＜１０％。     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.     

Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix,concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI‐TOF MS

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11‐mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI‐MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI‐MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI‐MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X‐100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave‐assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave‐digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.     

Controlled acid hydrolysis of colominic acid under microwave irradiation

The hydrolysis of colominic acid under microwave irradiation was studied and compared with traditional heating methods. The microwave irradiation has several advantages over the heating method in the hydrolysis of colominic acid: (a) products with higher degrees of polymerization are obtained, (b) less lactone byproducts are observed, and (c) the hydrolytic rate is much faster. These advantages are probably due to the microwave effect. Oligosialic acids as the products of the acid hydrolysis of polysialic acid with conventional heating methods were fully lactonized, especially under the conditions of higher temperature and stronger acid.     

Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis

A rapid heating method of hydrolysis by the use of microwave oven has been applied to amino acid analysis of proteins and peptides. This convenient method has been compared with the conventional 6 N HCl hydrolysis at 110 degrees for 24 h. The advantages of this new method are its expedition and the accurate and comparable results as compared to the tedious conventional technique. The method provides a rapid processing of multiple samples within minutes instead of days and inexpensive access to the important data of amino acid compositions of proteins by the commonly used microwave oven. The necessary change in the design of hydrolysis vials and the safety precautions accompanying this novel use of microwave acid-digestion method are also described.     

Determination of amino acids in foods by reversed-phase HPLC with new precolumn derivatives,butylthiocarbamyl, and benzylthiocarbamyl derivatives compared to the phenylthiocarbamyl derivative and ion exchange chromatography

New precolumn derivatizing reagents for analysis of amino acids by HPLC—butylisothiocyanate (BITC) and benzylisothiocyanate (BZITC)—reacted quantitatively with 22 standard amino acids and the amino acids in the acid hydrolysate of food and protein standard, bovine serum albumin (BSA), at 40°C for 30 min to yield butylthiocarbamyl (BTC) amino acids and at 50°C for 30 min to yield benzylthiocarbammyl (BZTC) amino acids. BTC and BZTC amino acids were successfully separated in 35 min on the reversed-phase Nova-Pak C18 column (30 cm × 3.9 mm, 4 μm). The optimum wavelengths for determination of BTC and BZTC derivatives were 240 nm and 246 nm, respectively. Analysis of the results obtained with BSA and food samples as BTC and BZTC derivatives showed good agreement with those determined as ion-exchange chromatography and data presented in the literature. The advantage of BITC reagent over the phenylisothiocyanate (PITC) and BZITC was that it had high volatility, so the excess reagent and by-products were easily removed in about 10 min, compared to about 1 h in the PITC and BZITC reagents. In the BTC and BZTC derivatives, cystine and cysteine were determined separately, but in the PTC amino acids derivatized with PITC reagent they were resolved into single peak.     

Partitioning behavior of amino acids in aqueous two-phase systems with recyclable volatile salts

As part of an ongoing research effort on aqueous two-phase systems (ATPSs) with volatile salts, this work describes the partitioning behavior of a series of amino acids, namely -serine, glycine, -alanine, -valine, -methionine, -isoleucine, and -phenylalanine, in these systems. The results show that amino acids partition in a similar way in polymer–volatile salt ATPSs and in traditional polymer–salt ATPSs. Increasing amino acid hydrophobicities lead to increasing partition coefficients. Moreover, the common linear relationship between the logarithm of the partition coefficient and the tie line length is observed here as well. Furthermore, the relation between relative partition coefficients and relative hydrophobicities of amino acids in the extraction systems investigated in this work is comparable to that in other extraction systems.     

Role of food partitioning in structuring the zooplankton community in mountain lakes

Trophic-niche differentiation is often cited as a main factor in structuring zooplankton assemblages, although field evidence for this is rarely presented. The study was based on a survey of 29 Pyrenean lakes with altitudes ranging between 1,875 and 2,990 m carried out during July and August 2000. Because of the oligotrophic nature of these lakes, we aimed to confirm that food partitioning is a major factor in shaping zooplankton assemblages. We analysed the amino acid composition of six cladocera and seven copepod species. A discriminant analysis showed that each species could be distinguished according to its amino acid composition. A negative relationship between amino acid differentiation and co-occurrence among the cladocera and cyclopoid copepod was observed. In contrast, calanoids did not show any relationship and were characterised by a high amino acid differentiation between species. As the differences in the amino acid composition among zooplankton species indicate distinct food sources, the relationship found indicates that trophic-niche differentiation plays a key role in determining the assemblage of these zooplankton communities. Therefore exploitative competition, either at present or in the past by driving co-evolutionary histories, has been a significant factor in structuring the cladocera and cyclopoid communities in these oligotrophic lakes.     

Isolation and quantitation of isotopically labeled amino acids from biological samples

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phtaaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Becuase the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken byb gas-phase acid hydrolysis (103% recovery after 5 h at 150°C: S.D = 3.5%, n = 14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 μl could be injcted, representing approximately 200 μl of deproteinized plasma. The methods were linear up to injection of 0.5 μmol of all amino acids (OPA: r²=0.995−0.999; FMOC: r²=0.992−0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50–500 nmol), was less than 3% above 100 mmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.     

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.     

Enzymatic hydrolysis of the Eisenia andrei earthworm: Characterization and evaluation of its properties

Some studies have carried out in order to retrieve proteins from the by-product of animal-processing industries. Earthworms are rich in protein and usually are used in animal feed. Thus, this study aimed to optimize the hydrolysis process of Eisenia andrei earthworms by employing Alcalase enzyme. Using the response surface methodology, we evaluated the following conditions: temperature, hydrolysis time, stirring speed, and enzyme/substrate ratio. The optimal conditions for the experimental design were determined through the analysis of the foaming and emulsifying properties, in vitro starch digestibility, and antioxidant activity. The results demonstrate that the highest degree of hydrolysis (i.e., 92%) was obtained under the following conditions: pH, 9.5; temperature, 25?°C; hydrolysis time, 2.25?h; stirring speed, 200?rpm; and enzyme/substrate ratio, 1.77%, using Alcalase enzyme. Evaluation of the amino acid composition under these conditions revealed higher concentrations of aspartic acid, glutamic acid, and leucine. The in vitro protein digestibility of the hydrolysate was approximately 73%. There were no significant improvements in either foam stability or emulsification after enzymatic hydrolysis. Additional studies on the antioxidant activity are required. This bioproduct could potentially serve as a promising supplementary food product.     

Easy Access to Enantiomerically Pure Nonproteinogenic Amino Acids

The protease from Bacillus licheniformis (alcalase) shows a remarkable broad substrate tolerance and high enantioselectivity against nonproteinogenic racemic amino acid derivatives. N‐acetyl protected amino acid esters of mono‐, di‐ or tri‐substituted phenyl alanines and even tert.‐leucine were hydrolyzed with high enantioselectivity. The obtained mixtures of (S)‐N‐acetyl amino acid and (R)‐N‐acetyl amino acid ester can easily be separated. The R‐ or S‐amino acids were obtained by acidic cleavage of the optically pure derivatives or the (R)‐ester was racemized by treatment with potassium t‐butylate.     

Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples

This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography–mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3 h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography–tandem mass spectrometry method.     

Amino acid distribution in some fungal melanins and of soil humic acids from Brazil

Summary Humic acids from four Brazilian topsoils of different origins and four soil fungal melanins, synthesized under two cultural conditions, were subjected to 6N HCl hydrolysis and their amino acid distribution patterns qualitatively and quantitatively determined. Both soil and fungal polymers showed similar patterns with aspartic acid, glutamic acid, glycine and alanine as the dominant amino acids. Some variations noted were more quantitative than qualitative, the similarities were more pronounced than differences, indicating that the fungal melanins may play a significant role in the formation of soil humic acid polymers. The humic acids of Brazilian soils had amino acid distribution patterns similar to those reported for humic acids of other tropical and temperate soils.     

Theophylline-7-acetic acid derivatives with amino acids as anti-tuberculosis agents

A series of amides were synthesized by condensation of theophylline-7-acetic acid and eight commercially available amino acid methyl ester hydrochlorides. Consecutive hydrolysis of six of the amido-esters resulted in the formation of corresponding amido-acids. The newly synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. The activity varied depending on the amino acid fragments and in seven cases exerted excellent values with MICs 0.46–0.26 μM. Assessment of the cytotoxicity revealed that the compounds were not cytotoxic against the human embryonal kidney cell line HEK-293T. The theophylline-7-acetamides containing amino acid moieties appear to be promising lead compounds for the development of antimycobacterial agents.     

海洋生物保健制剂中氨基酸的测定及营养评价

本文对以海参、鱼翅、鱼鳔等为主要原料,添加一些具有食疗作用的中药而制成具有不同功效的６种保健制剂,测定了其中的游离氨基酸及１８种常见氨基酸的含量。以ＦＡＯ／ＷＨＯ的氨基酸模式及化学评分,评价了这些保健制剂的蛋白质营养价值。限制性氨基酸主要是赖氨酸和（或）亮氨酸。这些制剂的氨基酸评分大多数都很高,有４种制剂的化学评分在９０分以上。     

Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques

A rapid method for the isolation of hydroxylysylpyridinoline and lysylridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pari chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemical within a short time.