气相色谱-质谱法测定中药沙苑子中的氨基酸

本文利用气相色谱一质谱(GC/MS)联用技术首次对中药沙苑子中的氨基酸进行了定性定量分析。沙苑子经盐酸水解后,对其氨基酸水解液进行羟基脂化和氨基酰化的衍生化反应,利用GC/MS法共测得15种氨基酸,由外标法测定了每种氨基酸的含量。由此得出沙苑子中氨基酸的总质量分数为4.23％。     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Amino acid analysis: determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive

All cysteine and cystine in a protein are derivatized during hydrolysis in hydrochloric acid containing 3,3'-dithiodipropionic acid. The resulting derivative can be separated from other amino acids and used for quantitation of cysteine plus half-cystine. A procedure is presented for accurate determination by ion exchange chromatography and postcolumn derivatization of all amino acids from acid hydrolysis of a protein, including the Cys-derivative.     

Debittering effect of Monascus carboxypeptidase during the hydrolysis of soybean protein

The actions of pepsin and the admixture of pepsin and Monascus pilosus carboxypeptidase 1 (MpiCP-1) on the hydrolysis of soybean protein were studied. The results showed that the pepsin hydrolyzate of soybean protein was much more bitter and contained relatively smaller amounts of total free amino acids than the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. In addition, hydrophilic and hydrophobic amino acids were present in almost equal proportions in the pepsin hydrolyzate, while mainly hydrophobic amino acids made up the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. These results suggest that MpiCP-1 suppresses and reverses the development of the bitterness taste that results from the pepsin hydrolysis of soybean protein by releasing mainly hydrophobic amino acids from the C-termini of the bitter components.     

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.     

Nitrotyrosine internal standard for amino acid analysis.

3-Nitrotyrosine is shown to be suitable as an internal standard for amino acid analysis. It is stable to acid hydrolysis, gives a color yield of 1.01 with ninhydrin which obeys Beer's Law, elutes in a position which is resolved from other amino acids on both Spinco and Durrum amino acid analyzers, and is commercially available. A method is also described for the preparation of a stable, pure, standardized stock solution of 3-nitrotyrosine.     

Conjugated Amino Acids in the Non-Cationic Fraction of Pea Seedlings

Many amino acids were liberated by acid hydrolysis of the non-cationic fraction of pea seedling extract, suggesting the existence of ninhydrin-negative conjugated amino acids. The non-cationic fraction was subjected to cellulose column chromatography and gel-filtration with Sephadex G-15 and was separated into 20 fractions which gave ninhydrin color after alkaline hydrolysis. Alanine and glutamic acid, the most predominant amino acids in the hydrolyzate of the non-cationic fraction, occurred in many fractions. These results indicated that alanine and glutamic acid existed in various conjugated forms. In one of the fractions, there was evidence suggesting the presence of a peptide containing equimolar amounts of alanine and glutamic acid.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

日立L-8900型氨基酸分析仪用缓冲液的研究

为立足于氨基酸分析仪所用缓冲液国产化,论证其可行性和可靠性,确定缓冲液的最佳配方,采用进口和自配两套缓冲液对各氨基酸的分离度、试剂的稳定性和定量分析的准确性进行比较。使用自配缓冲液,各氨基酸的分离度较好,自配试剂能够较长时间保持稳定,定量分析准确。结论:日立L-8900型氨基酸分析仪所用自配缓冲液保存时间长、分离效果好、定量准确、经济、适用,有一定的应用价值。     

The evaluation of the temperature effect and hydrolysis conditions for amino acid analysis

A systematic investigation of the optimal temperature and hydrolysis time for amino acid analysis has been carried out under various conditions. It is found that some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes and 110 C/24-72 hour hydrolysis can be achieved without loss of accuracy and performance in amino acid analyses of proteins and peptides. The effects of hydrolysis temperature and heating time on the recoveries of various labile and hydrophobic amino acids are exemplified in the hydrolysis of oxidized ribonuclease A, lysozyme and lens crystallin. The method provides a rapid processing of multiple samples within hours instead of days with the potential for the total automation of amino acid analysis starting from the preparation of protein hydrolysates.     

不同酶对草鱼蛋白功能特性及风味成分的影响

通过蛋白酶对草鱼进行酶解,对其酶解产物的功能特性和酶解液的风味成分进行研究。结果显示,风味蛋白酶、胰蛋白酶酶解产物的溶解性、热稳定性均优于胃蛋白酶酶解产物(P0.05)。经酶处理后鱼肉酶解液鲜甜味氨基酸占比增加,苦味氨基酸占比减少;利用SPME-GC-MS共检测出83种挥发性成分,以醛酮类和醇类为主,酶解处理后醇类的相对含量显著减少,醛酮类的相对含量则显著增加,酶解液具有独特风味。经胰蛋白酶、风味蛋白酶酶解后的草鱼肉酶解产物及酶解液均可作为食品基料应用于加工中。     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Isolation and primary characteristics of the common species-specific outer membrane protein of Rickettsia prowazekii

Common species-specific protein is isolated for the first time in the chromatographically pure state from the outer membrane of Rickettsia prowazekii, and its amino acid composition is determined. As revealed by chromatofocusing technique, the protein possesses pI 4.18 +/- 0.03. Three basic ninhydrin-positive compounds, differing from usual amino acids, were discovered in the protein hydrolyzate. Data suggesting the subunit structure of the isolated protein are presented.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

A fast and sensitive micromethod for the manual sequencing of peptides using O-phthalaldehyde as derivatizing reagent

A general procedure for the manual sequencing of peptides using the fluorogenic reagent O-phthalaldehyde (OPA) is described. The method can be applied in two different ways. One of them involves back hydrolysis of the anilinothiazolinones resulting from the Edman degradation of the peptide and subsequent detection of the free amino acids as OPA derivatives. The other is a subtractive analysis in which the amino acid composition of the remaining peptide is determined after each degradation cycle. The direct procedure can be coupled to the subtractive one in order to assure the accuracy of the sequence analysis. The method is fast and simple, and allows determination of 10 pmol of amino acid per cycle using standard reagents and instrumentation. Sensitivity can be greatly enhanced provided that ultrapure chemicals are employed. Small peptides (8-10 residues) were sequenced from 200 pmol sample, using a high-performance liquid chromatography assembly coupled to a fluorescence detector.     

不同来源仙草的氨基酸比较分析

采用盐酸水解法分析不同来源仙草的氨基酸组成及含量差异,结果表明供试仙草均含有17种氨基酸和7种必需氨基酸,氨基酸总量为4.75％～13.65％,17种氨基酸总量和7种必需氨基酸总量的高低顺序都是9＞10＞5＞8＞1 ＞6＞4＞3＞2＞7,均以9号仙草含量最高,7号仙草最低.各种氨基酸含量高低顺序相似,以谷氨酸、天门冬氨...     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amino acid and peptide ratio in protein hydrolysates for parenteral feeding

Using the method of amino acid analysis and routine methods of protein biochemistry, the ratio of amino acids and peptides in acid and enzyme protein hydrolyzates was determined. Depending on the production procedure, the hydrolyzates under study contained various amounts of free amino acids and peptides in which the number of amino acid residues varied from 2 to 7. Additional hydrolysis of these preparations by leucine aminopeptidase led to a decrease in the peptide content and to an increase in the amino acid content. This may have a beneficial effect on the quality of protein hydrolyzates.     

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.