Amino terminal analysis

Vera JC, Rivas Cl, Cortes PA et al (1988) Purification, amino terminal analysis, and peptide mapping of proteins after in situ postelectrophoretic fluorescent labelling. Anal Biochem 174 38 15... 

B 1. Study the details of the Edman and dansyl chloride methods for amino-terminal analysis of proteins. Discuss the advantages and disadvantages of each method. 

A fully carbamylated derivative of rabbit muscle phosphoglucose isomerase was utilized by James and Noltmann (1973), both for a quantitative amino-terminal analysis and for tryptic mapping experiments. Tryptic digestion of the derivative was restricted entirely to the arginyl residues. 

In order to characterize and confirm the analytic structure in the case of r-DNA technology-derived proteins or peptides, amino acid sequencing is the method of choice. Both the overall amino acid composition as well as N- and or C-terminal amino acid sequencing are useful and well-established tools in protein chemistry. Amino-terminal analysis reveals information about the primary structure, homogeneity and occurrence of cleavages in the polypeptide. The... 

Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but it does give amounts from which the percentages or ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-termuial end. 

The hybrid approach enhances the speed and efficiency of primary structure analysis and the range of proteins that can be sequenced. It also circumvents obstacles such as the presence of an amino-terminal blocking group or the lack of a key overlap peptide. Only a few segments of primary strucmre must be determined by Edman analysis. 

Microheterogeneity below specified level Polyacrylamide gel electrophoresis C- and N-terminal analysis. Amino acid composition... 

Figure 3 shows the final chromatogram and activity profile of purified alpha-endopsychosin. The first peak contained most of the PCP displacing activity as measured by its ability to inhibit 3H-PCP. An aliquot of the most active material was hydrolyzed in acid and the amino acid composition was determined using OPA detection. It was determined that the peptide contained approximately 26 amino acids, in close agreement with the molecular weight predicted by Sephadex gel filtration studies. N-terminal analysis revealed that the peptide was blocked at this site. The nature of this blockade is yet to be determined. Studies are under way to determine the amino acid sequence of the peptide. 

Shen CL, Scott GL, Merchant F, Murphy RM. Light scattering analysis of fibril growth from the amino-terminal fragment beta(l-28) of beta-amyloid peptide. Biophys J 1993 65 2383-2395. 

Gorman, J.J. (1984) Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis. Anal. Biochem. 160, 376. 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

Site-directed mutagenesis and the crystal structure analysis of a proteasome-inhibitor complex identified the amino-terminal threonine (Thrl) of Thermoplasma P subunits as both, the catalytic nucleophile and the primary proton acceptor (Seemiiller et al. 1995 Lowe et al. 1995). 

Consensus features were identified from comparison of sequence homologies of known sulfation sites 110139 and from in vitro sulfation of synthetic peptides using TPST-enriched membrane preparations 11-13 as well as from analysis of mutations. 14 These structural features mainly consist of a preferential type of charge distribution around the sulfation site, i.e. acidic residues on the amino-terminal side of the tyrosine, particularly in position 1. Full accessibility of the site to the TPST in surface-exposed loops and the absence of disulfide bridges in the proximity are also important for sulfation. 

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