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1.
Use of fluorescamine in the chromatographic analysis of peptides from proteins   总被引:10,自引:0,他引:10  
A routine procedure for the fluorometric analyses of peptides in the column chromatographic fraction has been described. Sensitivities of detection are 3–5 times higher in the direct fluorescamine method and 6–50 times higher in the method with alkaline hydrolysis than the conventional ninhydrin color method with hydrolysis (11).Reactivity of peptides with fluorescamine appears to depend mainly on the nature of amino acids occupying the amino termini; ?-amino groups of lysine residues in the peptides tested have been found not to contribute significantly in yielding fluorescence in the reaction.  相似文献   

2.
Amino acids were most likely available on the primitive Earth, produced in the primitive atmosphere or in hydrothermal vents. Import of extraterrestrial amino acids may have represented the major supply, as suggested by micrometeorite collections and simulation experiments in space and in the laboratory. Selective condensation of amino acids in water has been achieved via N-carboxy anydrides. Homochiral peptides with an alternating sequence of hydrophobic and hydrophilic amino acids adopt stereoselective and thermostable beta-pleated sheet structures. Some of the homochiral beta-sheets strongly accelerate the hydrolysis of oligoribonucleotides. The beta-sheet-forming peptides have also been shown to protect their amino acids from racemization. Even if peptides are not able to self-replicate, i.e., to replicate a complete sequence from the mixture of amino acids, the accumulation of chemically active peptides on the primitive Earth appears plausible via thermostable and stereoselective beta-sheets made of alternating sequences.  相似文献   

3.
Summary A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution3H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.  相似文献   

4.
Aluminum- and mild steel-binding peptides from phage display   总被引:4,自引:0,他引:4  
Using a phage library displaying random peptides of 12 amino acids on its surface, several peptides were found that bind to aluminum and mild steel. Like other metal-binding peptides, no obvious consensus motif has been found for these peptides. However, most of them are rich in hydroxyl-containing amino acids, serine or threonine, or contain histidine. For the aluminum-binding peptides, peptides with a higher number of hydroxyl-containing amino acids bind to the aluminum surface more tightly. For example, Val-Pro-Ser-Ser-Gly-Pro-Gln-Asp-Thr-Arg-Thr-Thr, which contains five hydroxyl-containing amino acid residues, was selected four-fold more frequently than a peptide containing only one serine, suggesting an important role for the hydroxyl-containing amino acids in the metal–peptide interaction.  相似文献   

5.
The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.  相似文献   

6.
Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.  相似文献   

7.
A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.  相似文献   

8.
Zaia DA 《Amino acids》2004,27(1):113-118
Minerals more readily adsorb amino acids with charged R groups than uncharged R groups, so that the incorporation of amino acids with charged R groups into peptides would be more frequent than for amino acids with uncharged R groups. However, 74% of the amino acids in the proteins of modern organisms contain uncharged R groups. Thus, what could have been the mechanisms that produced peptides/proteins with more amino acids with uncharged R groups than precursors with charged R groups? Should we expect the composition of amino acids adsorbed on minerals to be similar to those of present proteins? Was the adsorption of amino acids on minerals important for the origin of life? The lipid world offers an alternative view of origin of life. Liposomes contributed to elongation of peptides as well as select hydrophobic amino acids and peptides. These experiments could be showing the mechanism, which hydrophobic amino acids have been selected. However, liposomes have no influence on the stereoselectivity in the oligomerization of amino acids. In the present paper, several other mechanisms are also discussed that could produce peptides with a greater proportion of amino acids with uncharged R groups.  相似文献   

9.
Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.  相似文献   

10.
应用SignalP 3.0 对植物病原细菌Pseudomonas syringae pv. tomato DC3000菌株基因组中的全部5 615个ORFs进行了分析,确定其中679个ORFs所编码蛋白质的N-端有信号肽序列,其中已经命名并有注释的有107个ORFs。信号肽的长度以19 ~31 个氨基酸居多,其中最多的是23 个氨基酸的信号肽。具有信号肽的ORFs编码蛋白的长度大多为101~400 个氨基酸之间。同时,对组成信号肽的氨基酸种类作了系统的分析,发现组成信号肽的氨基酸中非极性氨基酸占48.54%,极性氨基酸占18.67%,带负电荷氨基酸占24.54%,带正电荷氨基酸仅占8.00%,出现最多的3种氨基酸依次为亮氨酸、丙氨酸和丝氨酸,最少的氨基酸是异亮氨酸,在切割位点-1端的氨基酸中83.211%均为丙氨酸,在切割位点后3位的氨基酸中最多的氨基酸也是丙氨酸。通过分析确定628个分泌类信号肽,36个信号肽具有RR-motif的保守区段,15个脂蛋白类信号肽,未发现Prepilin-like 信号肽和Bacteriocin and Pheromone信号肽。  相似文献   

11.
D-Amino Acids in Living Higher Organisms   总被引:2,自引:0,他引:2  
The homochirality of biological amino acids (L-amino acids) andof the RNA/DNA backbone (D-ribose) might have become establishedbefore the origin of life. It has been considered that D-aminoacids and L-sugars were eliminated on the primitive Earth.Therefore, the presence and function of D-amino acids in livingorganisms have not been studied except for D-amino acids in thecell walls of microorganisms. However, D-amino acids wererecently found in various living higher organisms in the form offree amino acids, peptides, and proteins. Free D-aspartate andD-serine are present and may have important physiologicalfunctions in mammals. D-amino acids in peptides are well knownas opioid peptides and neuropeptides. In protein, D-aspartateresidues increase during aging. This review deals with recentadvances in the study of D-amino acids in higher organisms.  相似文献   

12.
New alpha,alpha'-disubstituted amino acids with silylated side chains have been synthesized in racemic form. Starting from a Schiff base of glycine tert-butyl ester, a large variety of alpha,alpha'-dialkylated amino acids has been obtained, depending on the alkylating reagents. The application of a hydrosilylation methodology enabled the synthesis of the same unnatural amino acids in an enantiomerically pure form. The ability of these bulky amino acids to be incorporated into peptides by solution-phase methodology has also been demonstrated, since constrained silylated dipeptides have been synthesized. These new lipophilic building blocks could be useful and innovative in the design of peptaibol analogues.  相似文献   

13.
The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002  相似文献   

14.
The intensity of proteolytic processes and qualitative composition of autolysis products of the brain, liver and testicle tissues of young and old rats were studied. The gel-chromatographic analysis (Sephadex G-15 and G-50) revealed no considerable amount of high-molecular peptides (1500 Da and over) before and after autolysis. The measurement of the quantity of free amino groups in the gel-chromatographic fraction after the complete acid hydrolysis has confirmed that result. The low-molecular peptides and free amino acids, are the main products of the tissue autolysis. The intensity of proteolytic processes, determined by an increase in the amount of amino acids depends on the autolysis duration and age of animals. The total increment of amino acids in the brain and liver tissues of old animals for the first hour of autolysis has been higher by 102 and 219% as compared to young ones. The autolysis of testicles of the young and old animals after the first hour of incubation is characterized by the same intensivity. Such a regularity is not revealed when analyzing the same processes by the Lowry method.  相似文献   

15.
A new addition method is described in this study for calculating the partition coefficients of peptides. LogP and logD values of peptides are calculated by summing the contributions of the component amino acids. The final models are derived from a multivariate linear regression analysis of 219 peptides with known experimental data. The standard errors in a leave-one-out cross-validation are 0.23 and 0.24 log units for the logP and logD values, respectively. The predictive ability of the model is tested by an extra set of ten peptides, and the self-consistency of the model is further demonstrated by a new validation procedure called the evolution test. The parameters obtained in regression could be used as hydrophobicity scales for amino acids. The application of such hydrophobicity scales has also been discussed.  相似文献   

16.
The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.  相似文献   

17.
The syntheses of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH2 are described. The two peptides were prepared in solution by stepwise elongation using the DCC method with additives (HOSu or HOBt). The push-pull method was also used in the synthesis of Ac-Asp-Gly-Ser. A racemization test of the amino acids in this peptide has been performed.  相似文献   

18.
The carboxypeptidase Y-catalyzed reaction of acyl transfer of acylamino acid and peptide residues from the corresponding esters to ammonia and to amides of amino acids has been studied, and conditions for obtaining amides of amino acids and peptides with the yields up to 90% found.  相似文献   

19.
Chemisorption products of bifunctional amino acid vapours on the surface of silica and alumina have been studied by the method of infrared spectroscopy. On the basis of the analysis of spectral data it is supposed that heterogeneous polycondensation of amino acids with formation of peptides proceeds under these conditions. The supposition was confirmed by the study of products of interaction of amino acid vapours with silica and alumina by the method of fast atom bombardment mass-spectrometry. It is established that in contrast to alumina the condensation of amino acids into linear peptides on silica surface proceeds only at presence of at least small amounts of water. The most probable mechanisms of extending of peptide chains are proposed on the basis of obtained experimental data.  相似文献   

20.
Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N(2)-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.  相似文献   

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