Determination of Protein and Amino Acid

Chemical modifications of proteins have been carried out for a long time prior to any interest in the understanding of the chemical basis of the process. Early studies were motivated by the interest in quantitative determination of proteins and amino acids that conform its structure [104]. Intramolecular reactions occur naturally in posttranslational modifications such as disulfide bonding, glycosylation, or terminal residue cyclization. These modifications are relevant in structure-function relationships. They can produce conformational changes in order to switch between... 

Hurrell, R.F. and Finot, P.A. (1983). Food processing and storage as a determinant of protein and amino acid availability, in Nutritional Adequacy, Nutrient Availability and Needs, p. 135, J. Mauron, ed., Birhauser Verlag, Basel... 

Figure 8.2— Visualisation of proteins and amino acids by reaction with ninhydrin. This reagent, after a series of reactions, yields a Ruhemann scolouration. This is one of the reagents that can be used to determine the position of compounds that have migrated on the electrophoretic gel.

Jdnsson PG, Kvick A (1972) Precision neutron diffraction structure determination of protein and nucleic acid components. III. Crystal and molecular structure of the amino acid a-glycine. Acta Cryst B 28 1827 -1833... 

In the pioneering work on the occurrence and stability of proteins and amino acids in fossils, Abelson determined that thermally unstable amino acids such as threonine and serine were either much reduced or absent in fossils, whereas more stable amino acids, such as glycine and alanine, were still present (see ref. 5). The total concentration of amino acids decreases dramatically with time, von Endt and Erhardt reported their results concerning the differential chemical disintegration of amino acids in compact... 

The separation of amino acids has become a standard laboratory method of analysing protein hydrolysates, which are routinely prepared in the study of chemistry of proteins, and amino acid analyzers based on considerations discussed above are commercially available. Figure 8.5 is a schematic diagram of an amino acid analyzer using the ninhydrin reagent for quantitative determination. 

Proteins in foods such as milk have been the subject of much fluorimetric study involving measurement of the intrinsic fluorescence of tryptophan and tyrosine however, since proteins vary in their content of these amino acids, the fluorescence intensity also varies markedly from protein to protein and, for a single protein, with the experimental conditions. Also, the natural fluorescence of peptides is limited to peptide-containing tryptophans and tyrosines. However, the joint use of LC and derivatizing fluorogenic agents such as dansyl chloride, ninhydrin, fluorescamine, and o-phthaldialdehyde (OPA) has allowed the development of a number of methods for the determination of proteins, peptides, amino acids, and amines in food samples. 

Although the use of CE in food analysis is limited compared with LC, many applications have been reported in recent years in the study of food proteins and peptides. It has been shown that the sensitivity provided by CE using LIE detection for bovine whey proteins is twice as good as that provided by UV detection, under the same separation conditions. LIF detection has also been used together with affinity interactions to enhance the detection sensitivity in CE. For instance, an immunoassay detection method has been described using a polyclonal antibody marked with fluorescein to determine lactoferrin. CE-LIF has also been applied to the determination of amines and amino acids in several samples such as wine and milk products. 

Dimethylamino)ethanethiol DMAET-capped CdTe NPs were employed in the construction of aptamer-based biosensing system for proteins [109]. Highly efficient and sensitive determination of alkaloids and amino acids using CE in conjunction with sequential light-emitting diode-induced fluorescence (LEDIF) and ECL method was carried out. In the CE-LEDIF-ECL system, the ECL detector was located in the outlet of the capillary, while the LEDIF detector was positioned 12 cm from the outlet [110]. Ru(bpy)3 in MWCNTs/Nafion composite membrane was immobilized for an ECL method for reduced nicotinamide adenine dinucleotide (NADH) with a detection limit of 8.2 x 10 M [111]. 

Urea is synthesized from the ammonia derived from protein, polypeptide, and amino acid catabolism. Consequently, the amount of urea in the blood is partly determined by protein and amino acid metabolism. Therefore, in considering urea production, the source of the ammonia and the biosynthesis of urea must be discussed. The ammonia is derived mainly from amino acid breakdown. In liver, the amino acid pool is derived from the breakdown products of ingested and endogenous proteins and from amino acids synthesized de novo. 

A. General Procedures for Determination of Proteins and Amino. - cids. B. General Procedures for Preparation of Peptides and Amino Acids. C. Specific Procedures for Isolation and Determination of Individual Amino Acids. 

In situ and in vitro methods to determine intestinal digestion of protein and amino acids in ruminants... 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

One of the important purposes for the study of the direct electron transfer of protein is to construct the mediator-free protein-based biosensors. These biosensors can determine many small molecules like H202, 02, NO, nitrite, small organic peroxide, and so on. They also can determine glucose, alcohol, and amino acids by... 

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PILL tube (20 cm X 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4+ and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36],... 

The history of molecular biology has been a history of technological developments for determining the primary and tertiary structures of protein and nucleic acid molecules. Once the molecular structure is known, it provides clues to molecular functions. This is the principle of the structure-function relationship. Based on this principle the analysis of the amino acid sequence is performed to decipher the functional information from the sequence information. The analysis usually involves detection and prediction of empirical sequence—function relationships with additional consideration of known or predicted three-dimensional (3D) structures. Thus, the process can be represented schematically as ... 

Kataoka H. 1997. Selective and sensitive determination of protein and non-protein amino acids by capillary gas chromatography with nitrogen—phosphorus selective detection. Biomed Chromatogr 11 154. 

Gas-liquid chromatography used for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins was described in 1976 by Davy and Morris. Based on carboxypeptidase A digestion of the protein, the partially digested protein was removed and the amino acids released after known time intervals were analyzed by quantitative gas-liquid chromatography. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins. Thus the amino acid sequences were determined. 

The second part of the proof of colinearity of DNA and protein sequences was the determination of the complete amino acid sequence of tryptophan synthase and peptide mapping (Chapter 3) of fragments of the mutant enzymes. From the peptide maps it was possible to identify altered peptides and to establish the exact nature of the amino acid substitutions present in a variety of different tryptophan auxotrophs. When... 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Eriksson, S. and J. Sjoquist Quantitative determination of N-terminal amino acids in some serum proteins. Biochim. Biophys. Acta 45, 290 (1960). 

The present investigation was undertaken to determine if the methionine sulfoxide found by several investigators in insect tissue is a normal constituent of the amino acid pool and of protein. The amino acid composition of cockroach protein was simultaneously determined both qualitatively and quantitatively, since such data are available for few species of insects. 

Most doctors use the plasma concentrations of creatinine, urea and electrolytes to determine renal function. These measures are adequate to determine whether a patient is suffering from kidney disease. Protein and amino acid catabolism results in the production of ammonia, which in turn is converted via the urea cycle into urea, which is then excreted via the kidneys. Creatinine is a breakdown product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body (depending on muscle mass). Creatinine is mainly filtered by the kidney, though a small amount is actively secreted. There is little to no tubular reabsorption of creatinine. If the filtering of the kidney is deficient, blood levels rise. 

One of the first applications of HPLC in the clinical field was the quantitation of theophylline in asthmatic infants. This highly accurate measurement was an important test because of the very low amount of sample required and the accuracy of the determination (see Fig. 1-10). More recent clinically related HPLC separations include drugs and drug metabolites, neurochemicals and their metabolites, histamines, thyroid hormones, and enkephalins. The earliest bioresearch applications of HPLC included the determination of peptides, proteins, and amino acids. Application of HPLC to the analysis of these compounds remains important, as indicated by the rapid growth in references (Fig. 1-11). Bioresearch remains one of the most rapidly expanding growth areas of LC. 

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