Proteins 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. 

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. 

Dinitrofluorobenzene (86, X = F) is, because of its reactivity, much used for tagging the NH2 group of terminal amino-acids in protein end group analysis. Once it has reacted with the NH2 it is very difficult to remove again and will thus withstand the subsequent hydrolysis of the protein to its constituent amino-acids. 

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. 

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. 

You have isolated and purified a new enzyme (E) which converts a single substrate (S) into a single product (P). You have determined Mr by gel filtration as 46,400. However, in SDS gel electrophoresis, a molecular mass of 23 kDa was indicated for the single protein band observed. A solution of the enzyme was analyzed in the following way. The absorbance at 280 nm was found to be 0.512. A 1.00 ml portion of the same solution was subjected to amino acid analysis and was found to contain 71.3 nmol of tryptophan. N-terminal analysis on the same volume of enzyme revealed 23.8 nmol of N-terminal alanine. 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

Maly et al. (1980) irradiated E. coli 50 S ribosomes and found that protein L4 was specifically crosslinked to the 23 S RNA. Again a 1 1 adduct was isolated, and after pepsin digestion, label was found in two peptides. Amino acid analysis showed that the peptides were similar and that a Tyr residue (residue 45 of L4) was altered. In one peptide the altered residue was N-terminal and in the second peptide the residue was the fifth and modified differently from that in the first peptide, in such a way that Edman degradation was blocked. 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

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