Amino acid analysis protocols

Hughes, GJ. and Frutier, S. 1990 Amino acid analysis protocols, possibilities, and pretensions. In Fini, C, Floridid, A., Finelli, V.N. and Wittman-Leibold, B., eds., Laboratory Methodology in Biochemistry Amino Acid Analysis and Protein Sequencing. Boca Raton, FL, CRC Press, Inc. 44-61. 

Cooper C, Packer N, Willims K, eds. Amino Acid Analysis Protocols. Totowa NJ Humana Press, 2001 1-265. 

Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams,... 

Cooper, C., Packer, N., Williams, K., Eds. Amino Acid Analysis Protocols, Humana Press Totowa, NJ, 2001. Smith, B.J., Ed. Protein Sequencing Protocols, Humana Press Totowa, NJ, 2003, 111-194. 

The degree of glycosylation of the glycopolypeptides was estimated via H NMR, MALDI-TOF, and periodate assay (Glycoprotein Carbohydrate Estimation Kit (Pierce, Rockford, IL). H NMR spectra were acquired on a Bruker DRX-400 NMR spectrometer under standard quantitative conditions at 25 °C, and the standard protocols for the periodate assay were as described by the manufacturer. Comparison of sample solutions to a calibration curve of proteins of known carbohydrate content, combined with quantitative amino acid analysis of the samples, permitted estimation of the degree of substitution of the glycopolypeptides. 

Amino acid analysis, determination of amino acid composition of a peptide by complete hydrolysis followed by the quantitative analysis of the liberated amino acids. For hydrolysis, numerous chemical and enzymatic protocols are known. Based on the pioneering studies of Stein and Moore, amino acid analysis has been automated. Nowadays, instmments are in use for quantitative amino acid analysis which are based on partition chromatography, such as HPLC and gas-liquid chromatography [S. Blackburn, Amino Add Determination, M. Dekker, New York, 1978 ... 

Peptide hydrolysis, complete hydrolysis of peptides and proteins for amino acid analysis or the production of individual amino acids from the peptide or protein hydrolysate. For this purpose, numerous chemical and enzymatic protocols are known, but none of these procedures alone is fully satisfactory. Besides hydrolysis with 6 M hydrochloric acid at 120 °C for 12 h, or with dilute alkali (2-4 M NaOH) at 100 °C for 4-8 h, mixtures of peptidases can also be used for complete peptide hydrolysis. Restricted or limited peptide hydrolysis ( peptide cleavage) is important for - sequence analysis and peptide mapping. 

Methodology for determining the amino acid composition of proteins. The consultation concluded that modem amino acid analysis can provide data with repeatability within a laboratory of about 5% and reproducibility between laboratories of about 10%. It recommended that this variability be considered acceptable for the purposes of calculating the amino acid score. To achieve such results requires careful attention to many aspects of the protocols, including replicating the complete analytical procedure [118]. 

Figure 1. Protocol 1 standard amino acid analysis. A calibration mixture containing 1.0 nmol of each amino acid was injected onto a 0.40 x 13 cm bed of Dionex DC-5 A cation-exchange resin. Four discrete buffer solutions were pumped through the column at 18 ml/hr to achieve the indicated separation. Column temperature 45°C changed to 65°C at 12 min. Eluent pump pressure was 55 atm at 45°C. o-Phthalaldehyde reagent was added to the column effluent to produce fluorescent derivatives, which were continuously monitored using a fluoro-meter and strip chart recorder.

Figure 3. Protocol 2 of amino acid analysis. If the initial column temperature is lowered to 30°C, asparagine and S-carboxyethylcysteine can be resolved. Note change in relative elution positions of aspartic acid, threonine, and serine as a result of lower column temperature.

Figure 4. Protocol 3 of amino acid analysis. If the pH of eluent C is lowered to 6.50, methylated histidines as well as mono- and diiodotyrosine can be resolved. Other conditions of analysis are as in Fig. 1.

Figure 5 Protocol 4 of amino acid analysis. In this case, the pH of eluent D is lowered to 9.0 in order to effect separation of N -trimethyllysine. Note that this increases the total analysis time (compare Fig. 1).

Figure 6. Protocol 5 of amino acid analysis. Separation of methionine sulfoxide and methionine sulfone as well as 3- and 4-hydroxyproline (not shown) can be accomplished by the introduction of an eluent before the normal A eluent. This pre-A solution has a pH of 2.86 and contains 6% n-propanol. Changeover to the normal A eluent occurs after 12 min. Other conditions as in Fig. 1.

Figure 7. Protocol 6 of amino acid analysis. Separation of isodesmosine and desmosine is achieved by inserting an eluent with pH 6.0 between the normal B and C solutions. This pre-C eluent breaks through immediately after phenylalanine and continues until after desmosine is eluted. Normal programming continues thereafter.

A well-established conventional protocol to locate disulfide bonds in proteins includes cleaving a protein between the half-cysteinyl residues in a manner that leaves the disulfide bonds intact, isolating the cysteine-containing peptides, and performing either amino acid analysis or sequence analysis [2]. Alternatively, the disulfide bonds are reduced sequentially to thiols and alkylated with radiolabeled iodoacetic acid [1,3]. The completely reduced protein is cleaved enzymatically, and the disulfide bonds are detected by identifying the radiolabeled peptides. 

In this protocol, amino acid analysis on the submicrogram level of proteins and peptides electroblotted onto PVDF as well as in solution is described. 

P. Jandik. C. Pohl, V. Barreto and N. Avdalovic, Anion exchange chromatography and integrated amperometric detection of amino acids. In C. Cooper. N. Packer and K. Williams (eds.), Mahods in Molecular Biology, Vol. 159 Amino Add Analysis Protocols, Humana Press, Inc. Totowa, NJ, USA 2001. 

Bhushan (1991) on amino acids and their derivatives their new review includes references through 1994. Jain (1996) has reviewed studies on the applications of TLC to amino acid analysis of biological fluids and tissues. Such analyses are important in making diagnoses of inborn errors of amino acid metabolism. It was noted by Jain (1996) that TLC has proved useful to screen and quantify abnormal amounts of free amino acids in blood and urine samples. Shalaby (1996) in his chapter on TLC in food analysis has provided some protocols on amino acid analysis following the hydrolysis of protein samples. Fried and Haseeb (1996) in their chapter on TLC in parasitology have provided some information on the analysis of free pool amino acids from tissues of parasites and from the hemo-lymph of mosquitoes infected with Plasmodium (the malaria organism). 

The standard protocol for analysis of the amino acid composition of proteins is discussed in Section 5.1. Results of such analyses allow the researcher to anticipate which methods of polypeptide fragmentation might be useful for the protein. 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

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