Amino-acid analyser production

The product quaUty considerations for nonphotosynthetic microorganisms are similar to those for algae. Tables 6 and 7 present composition and amino acid analyses, respectively, for selected bacteria, yeasts, molds, and higher fungi produced on a large pilot-plant or commercial scale. Table 8 summarizes results of proteia quaUty and digestibiUty studies. 

LPC Product Quality. Table 10 gives approximate analyses of several LPC products. Amino acid analyses of LPC products have been pubhshed including those from alfalfa, wheat leaf, barley, and lupin (101) soybean, sugar beet, and tobacco (102) Pro-Xan LPC products (100,103) and for a variety of other crop plants (104,105). The composition of LPCs varies widely depending on the raw materials and processes used. Amino acid profiles are generally satisfactory except for low sulfur amino acid contents, ie, cystine and methionine. 

Successful and reproducible separations require a steady buffer flow rate and this is achieved with either a constant pressure or a constant displacement pump. These pumps are designed to deliver a constant rate of fluid independent of the resistance to the flow and recent developments in pump design permit the production of a precise and pulseless flow this has contributed towards the increased analytical precision and sensitivity that can now be achieved with amino acid analysers. The choice of flow rate is dependent upon the type of resin, the dimensions of the column and overall design of the instrument and this varies between models. 

Cys(Acm)U5]-endothelin I [H-Cys(Acm)-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys(Acm)-His-Leu-Asp-Ile-Ile-Trp] (22 0.41 pmol) was treated with silver(I) trifluoroacetate/DMSO/ 1M HCI in an analogous manner to that described in Section 6.1.1.1, The HPLC elution pattern of the crude peptide showed two peaks, with retention times identical to those of endothelin-I and an isomer in a ratio of 8 1. Each product was purified by HPLC to yield endothelin-I (23) and its isomer (7.5% yield 62%) these products were characterized by HPLC, FAB-MS, and amino acid analyses. 

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). 

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). 

Additional evidence for a bacterial contribution to HMW DOM proteins comes from molecular-level analyses of dissolved amino acids. Hydrolysis of HMW DON releases 11-29% of the nitrogen as amino acids (McCarthy et al., 1996). Specific amino acids include common protein amino acids, as well as /3-alanine and y-aminobutyric acid which are nonprotein amino acid degradation products. The distribution of amino acids is similar to that of fresh plankton cells, suspended particulate matter, and total dissolved amino acids. However, stereochemical analyses show HMW DOM amino acids to be elevated in the D-enantiomer, with d/l ratios for alanine, aspartic acid, glutamic acids, and serine ranging from 0.1 to 0.5 (McCarthy et al., 1998). Racemization of phytoplankton-derived L-amino acids is too slow at ocean temperatures to yield such high D/L ratios, but bacteria can synthesize D-amino acids, and it is likely that the D-amino acids in HMW DOM result from bacterial bioploymers rich in these particular amino acids. The high dA ratios of some amino acids and the abundance of amide nitrogen in HMW DOM N-NMR spectra led McCarthy et al. (1998) to... 

Finally, treatment with reagents such as tetranitromethane, may lead to products in which the expected tyrosine derivative is absent. The extent of formation of such derivatives is difficult to assess from amino acid analyses. 

The amino acid analyses of food products report cystine instead of cysteine. Cystine is an amino acid that is formed from the oxidation of two molecules of cysteine. 

A similar problem associated with synthetic peptides of chain length > 5 amino acid residues is the identification of side reactions. Those involve substitutions on imidazole-, phenol- and indole moieties of histidine, tyrosine, and tryptophane as well as conversions, transamidation, and cyclizations of aspartic and glutamic side chains. As long as structural variations are stable under the reaction conditions of an Edman degradation, they can be detected from the proper phenylthiohydantoines in combination with H-NMR- and mass spectrometry. Quantitative amino acid analyses of impure peptides after acidic total hydrolysis do not indicate those structural deviations between main product and contaminations. 

Since biological value is dependent primarily upon essential amino acid constitution, it would seem logical to assess the nutritive value of a protein by determining its essential amino acid constitution and then comparing this with the known amino acid requirements of a particular class of animal. Application of modern chromatographic techniques coupled with automated procedures allows relatively quick and convenient resolution of mixtures of amino acids. However, the acid hydrolysis used to produce such mixtures from protein destroys practically all the tryptophan and a considerable proportion of the cystine and methionine. Tryptophan has to be released by a separate alkaline hydrolysis, and cystine and methionine have to be oxidised to cysteic acid and methionine sulphone to ensure their quantitative recovery. Losses of amino acids and the production of artefacts, which are greater with foods of high carbohydrate content, are reduced if the hydrolysis is carried out in vacuo. Evaluations of proteins in terms of each individual amino acid would be laborious and inconvenient, and several attempts have been made to state the results of amino acid analyses in a more useful and convenient form. 

The reaction is the basis of various methods for the determination of amino acids since it is possible to measure (1) the CO2 produced, (2) the NH3 produced and (3) the colour intensity obtained when the liberated ammonia reacts with a further molecule of ninhydrin to produce a purple compound which can be assayed photometrically. This is the method by which amino acids are estimated using an amino acid analyser. The imino acids (proline and hydroxyproline) give yellow products instead of a purple one. Amino acids give a strong reaction with ninhydrin, but proteins and polypeptides, which contain far fewer free amino groups, give a much weaker reaction. 

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. 

---