Amino acid analyser analysis

The question was whether impurities were present in the samples analysed (Bada et al., 1983). In a more recent publication, Cronin and Pizzarello (1997) reported amino acid analyses using Murchison material in which an excess of L-enantiomers was present. Contamination with terrestrial biological material can be ruled out, as the amino acids in question are not proteinogenic a-methylamino acids, which occur either extremely seldom or not at all in terrestrial life forms, were detected. GLPC/mass spectrometry (MS) analysis gave the following enantiomeric excess (ee) values ... 

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. 

Although this technique is relatively straightforward and automated amino acid analysers are commercially available, it is subject to a number of disadvantages that limits its usefulness in bi-opharmaceutical analysis. These include ... 

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

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 identification and quantitation of the individual amino acids in a mixture is often required in metabolic studies and investigations of protein structure. The use of thin-layer chromatography or electrophoresis may be adequate to indicate the relative amounts and number of different amino acids in a sample but the use of gas-liquid chromatography or an amino acid analyser is essential for quantitative analysis. 

For the analytical characterization of sulfated tyrosine peptides, spectroscopic methods as well as amino acid analysis and, more recently, mass spectrometry are employed. In Table 2 the spectroscopic data of tyrosine 0-sulfate are compared to those of the related sulfonic acid derivatives as possible byproducts in the chemical sulfation of the tyrosine or tyrosine peptides.[361 In the course of the synthesis of tyrosine 0-sulfate peptides and, particularly in the final deprotection step, desulfation may occur which limits the characterization of the final compounds in terms of quantitative identification of the tyrosine 0-sulfate. This is achieved by amino acid analyses of basic hydrolysates of the sulfated tyrosine peptides or preferably by analyses of the enzymatic hydrolysates with aminopeptidase M or leucine-aminopeptidase. 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

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. 

You have isolated a tetrameric NAD+-dependent dehydrogenase. You incubate this enzyme with iodo-acetamide in the absence or presence of NADH (at 10 times the Km concentration), and you periodically remove aliquots of the enzyme for activity measurements and amino acid composition analysis. The results of the analyses are shown in the table. 

To obtain data on the heterogeneity of the glycopeptide fraction of microbubble surfactant, comparative amino acid analyses were performed on two of the major peaks obtained from gel filtration. From the ratio of absorbances at 230 and 280 nm (ref. 265) and the elution profile shown in Fig. 5.3, it appeared that peaks I and III would differ the most in amino acid composition and, therefore, these two peaks were selected for amino acid analysis. Peak I was sufficiently large to be divided into three equal aliquots and peak III into two equal aliquots for automated analysis. Peak II, which eluted closest (Fig. 5.3) to the dominant peak I and presumably was most similar in molecular composition to this large initial peak, was analyzed separately by HPLC for carbohydrate content. 

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. 

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. 

Experimentally, the first step in amino acid analysis, whose origins go back to the work of Stein and Moore in 1948, is hydrolysis of the protein. The protein sample should not contain too much salt, detergent or other additives, and dialysis against a dilute buffer or even water is recommended as a first step. The dialysed solution is then dried in vacuo the quantity needed depends on the sensitivity of the amino acid analyser (typically 1-10 nmol, with older forms of apparatus needing 10- tolOO-fold larger size samples). The dried sample is then subjected to hydrolysis 6 M HQ at 150 °C for 6 h, or 125 °C for 24 h, or 110 °C for 48 h. To exclude oxygen, 0.02 % (v/v) 2-mercaptoethanol and 0.25 % (w/v) phenol are added to the hydrolysis. With the most sensitive amino add analysers currently available, it is possible to analyse protein samples recovered from polyacrylamide gels and blotted onto PVDF-membranes. 

Supporting evidence for this proposal was obtained from amino acid analyses which demonstrated that glycine was the only typical or non N-methylated amino acid present in 4ab and Sab. Marfey analysis of the acid hydrolysates of 4ab, Sab and majusculamide C (6) confirmed that 4ab and Sab differed from majusculamide C (6) in possessing an N-methyl-L-alanine residue rather than an L-alanine residue. Moreover, 4ab and Sab contained an N-methyl-L-leucine residue rather than an N-methyl-L-isoleucine residue,... 

Amino acid analysis remains an indispensable tool in a variety of biological research and development fields, e.g. the biochemical study of proteins, quality control in biotechnology and nutrition, and in clinical analyses. The classic chromatographic technologies will be for the foreseeable future the major quantitative tools for amino acid analyses. The techniques are deceptively difficult and there remains a need to standardize techniques for good quantitation. 

Studies of KH clearly indicate complexity that is only partially resolved (49). DiflFerential staining reveals small, dense, homogeneous particles within amorphous KH masses, usually associated with tono-fibrils (32, 48, 49). Amino acid analyses of supposed KH materials show at least three distinctive patterns (see Table I). The amorphous material of Tezuka and Freedberg (72) has much less proline and cystine than KH studied by Matoltsy (51). Other workers have associated histidine with KH in granular cells (48, 49, 76). Tezuka s histidine values (72) fall between those of Matoltsy (51) and Hoober (76) and conceivably represent an analysis of mixed components. UgeFs bovine material is a nucleoprotein that may be either a ribosomal product or still another KH component (71, 77). 

HPLC has been used particularly in the analysis of amino acids and a large number of amino acid analysers are available commercially. For example, the use of a high-pressure, single-column amino acid analyser that can give a complete analysis of a protein hydrolysate in 42 min was described by Benson [254]. 

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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