Amino-acid analyser determination

Nitrous acid-induced mutations in tobacco mosaic virus (TMV) have contributed to the derivation of the genetic code (see Table 8.1) through amino acid analyses determining replacements in the coat protein (2). Since only C- U, G- A or A->G transitions are possible in the case of a nitrous acid point mutation, the number of possible replacements for a given amino acid is small. Thus, an Ala Val transition corresponds to a change of the codon GCN to GUN, whereas an Ala- Thr transition must be due to a change in the first letter GCN- ACN (See Chapter 8, Table 8.1). 

The classic work of Dawson and Pritchard [264] on the determination of a-amino acids in seawater uses a standard amino acid analyser modified to incorporate a fluorometric detection system. In this method the seawater samples are desalinated on cation exchange resins and concentrated prior to analysis. The output of the fluorometer is fed through a potential divider and low-pass filter to a comparison recorder. 

This latter method is mainly used when a single substance in a sample is being determined but where the analysis involves the quantitation of many or all of the components of the sample, e.g. in an amino acid analyser, the former method is the more suitable. 

The aqueous reagent is stable at room temperature and the reaction proceeds quickly without requiring heat. The method is approximately ten times more sensitive than the ninhydrin method and is particularly useful when the quantitation of many amino acids is being carried out using amino acid analysers or HPLC. However, the fluorescent yield of individual amino acids varies and fluorescence values must be determined for quantitative work in the same manner as the colour values for ninhydrin. 

Sowden et al. [4] also did detailed amino acid and amino sugar analyses of the soils from the different dimatic regions. The following amino acids were determined acidic amino acids aspartic and glutamic acids basic amino acids arginine, histidine, lysine and ornithine neutral amino adds, phenylalanine, tyrosine, glycine, alanine, valine, leudne, isoleudne, serine, threonine, proline and hydroxyproline ... 

When air oxidation of the reduced p-conotoxin GIIIB (18) was carried out in 0.1 M NHtOAc buffer (pH 7.5) at 0.01 mM peptide concentration and at 10 °C, three major products, isomers 15,16, and 17 were produced after 40 hours in a ratio of 1 4 3 (Figure 2). 86 The disulfide structures of each isomer were determined by enzymatic digestion followed by amino acid analyses, mass spectrometry, sequence analyses, as well as by the synthetic approach (Scheme 10). 

There are two major categories for amino acid analysis (a) free amino acid analysis and (b) determination of total amino acid content. The total amino acid content includes contributions from the free amino acids and the amino acids that are originally protein bound. These protein-bound amino acids must first be liberated before chromatographic analysis. This necessitates a more extensive, and problematic, sample preparation. Because the sample preparation procedures are so disparate, it is convenient to address these two categories of amino acid analyses separately. It should be noted that while the sample preparations for these analyses are quite different, both utilize essentially the same chromatographic techniques for the second stage of amino acid analysis. 

Crude protein and total lysine plus furosine Total nitrogen was determined by macro Kjeldahl digestion and protein estimated as N x 6.25. Total lysine values were obtained from conventional amino acid analyses carried out on 500-mg samples following digestion with 800 ml of 6M HC1 under reflux by use of a Biotronic LC 6000 or Kontron Liquimat III amino acid analyzer. Furosine was determined in the same way using 300 ml 7.8 M HC1 as described in ( 6 ) with an amino acid analyzer ( 7 ). 

Kovatcheva [12] has described a method using an automated amino acid analyser for the determination of S-methylmethionine in cabbage, kohlrabi, celery and sweetcorn. The plant sample is first homogenised with 0.1N hy-... 

In addition to analyzing a protein for characteristics dictated by its specific nature, routine analytical assays are performed namely, amino acid analyses and amino- and carboxy-terminal sequencing. If necessary, disulfide assignments are made. A search is ordinarily conducted for oxidations and/or deamidations. In the case of recombinant proteins particular attention is paid to detecting signal sequences or proteolytically degraded species. In contrast, deletions and chemical modifications can be a consequence of proteins prepared by chemical synthesis. Lastly, tests are applied to determine if the protein is correctly folded into its native three dimensional structure. In addition, with recombinant proteins, sensitive immunological procedures are performed to ensure that host cell proteins have not been copurified with the protein of interest. 

An amino acid analyser has been adapted for the determination of CML and CEL.387... 

With these newer methods of protein separation and amino acid analysis he prepared serum protein fractions by serial salting out with ammonium sulfate and by the Sober and Peterson DEAE cellulose columns (42), using the sera of reptile, fowl, and mammalian blood. Some of the amino acid analyses were carried out by the automatic amino acid methods of Hirs, Moore, and Stein (18). Fortified with this plethora of data, Block now had the opportunity to re-examine not only the ratio of the basic amino acids, but at least 12 amino acids in a variety of protein fractions prepared by at least two different procedures. With the aid of a statistician he determined the significance of the constancy of the molar ratios of pairs of amino acids and found that in spite of the marked variation of the absolute amounts of an amino acid, the molar ratios of certain pairs remain relatively constant among the numerous protein components of animal sera. 

Pentadin is a sweet-tasting protein that was isolated from the fruit of Oubli (P bmzzecma Baillon), a climbing shrub that is native to West African countries. Pentadin s molecular mass is estimated to be 12kDa. It is reported to be 500 times sweeter than sucrose on a weight basis. The primary structure has not yet been determined, although amino acid analyses were carried out.91... 

In conclusion, nitrogen/carbon ratios combined with quantitative amino acid analyses could determine the level of impurities that may co-exist with fossil bone collagen and could help in selecting the optimum method of collagen separation. An extraction method may be successful in some cases but could fail to remove the impurities from bone collagen in other samples. Chemical analysis of the impurities and their radiocarbon dates also should be obtained. 

Amino acid analysis is the determination of the composition of the 20 component amino acids in a protein (Ozols 1990). It is an essential part of characterising a protein, and information about composition can be used in a wide variety of different ways, not least to corroborate information from other sources about the purity of a protein only single species will have a whole number ratios for the molar proportions of the individual amino acid residues. Quantitative amino add analyses can be used to determine the concentrations of proteins with high sensitivity (pg range) and accuracy ( 10 %), although specialised amino acid analysers are needed to do this. 

Barnett for molecular weight determination, Blanche Hall for amino acid analyses, and Betty W. Li for disulfide bond analyses. 

Anderson et al. (1963) have described a more comprehensive system for nucleotide analysis. Their system has been recommended for adaptations of some amino acid analysers. A single column 0.9 X 160 cm of Dowex-1 ( x8, 200- 00 hydraulically fractionated to about 60 p particle diameter) washed in acid and alkali was packed in sections in 0.15 M sodium acetate pH 4.4. The sample, vol 0.5-1.5 ml in buffer, was eluted by a 1.4-1 linear gradient from 0.15-3 M sodium acetate at a constant pH (4.4), flow-rate (1 ml/min) and temperature (40°C). The first few peaks were extremely sharp and a lower flow rate could be used here. Good separations of quite complex mixtures were obtained in 28 hr. The triphosphates, UTP, ATP, GTP could be separated more quickly (in 6 hr) on a 0.9 x 50 cm column of the same resin eluted with a 1 1 linear gradient from 0.5 M sodium acetate, 0.25 M NaCl to 1.0 M sodium acetate, 0.5 M NaCl pH 3.6 at 1.7 ml/min at room temperature. A freshly packed column was used for each determination in both cases. 

Rather than resort to purely empirical selection of suitable values of ni and p(/Cint)< for equation 1 it is more usual to begin by fitting experimental data with values of m chosen to conform with the numbers of prototropic groups determined by several more direct and specific methods of examination of titration data. Even where the theoretical analysis of a titration curve is not attempted and exact values of p(Ki t), for each type of group are therefore lacking, the numbers of groups so determined may furnish valuable clues to the internal structure of the protein, especially when they are compared with the results of amino acid analyses. 

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