Total hydrolysable amino acid

Early studies looked at both dissolved free and combined amino acids (DFAA and DCAA, respectively) in total DOM, where HMWDOM proteins are a subset of the DCAA fraction. Lee and Bada (1975) first reported DFAA concentrations in the range of 40—50 nM in surface Pacific waters and total hydrolysable amino acid (THAA = DFAA + DCAA) concentrations that were often 10 times higher. To date, the observed range of TFIAA is approximately 250—650 nM in surface waters (Dittmar et al., 2001, 2004 Hubberten et al., 1995 Yamashita and Tanoue, 2003). Lee and Bada (1975) observed an approximate three-fold decrease in THAA below the euphotic zone (at these depths DFAA became negligible). This trend was also seen in more recent studies where amino acid concentrations decreased to between 160 and 360 nM in mid-depth waters (Hubberten et al., 1995 Yamashita and Tanoue, 2003). McCarthy et al. (1996) found that HMWDOM-DCAA concentrations were 178 and 278 nM in surface waters of the Sargasso Sea and North Pacific Ocean, respectively. 

Dissolved amino acids are commonly divided into two pools that must be analyzed separately DFAA exist as individual monomers in solution, while DCAA are defined operationally as additional amino acids liberated by acid hydrolysis. DCAA are thus presumably present mostly as polypeptides, a supposition supported in at least the high molecular weight (HMW) fraction by N-nuclear magnetic resonance (NMR) spectroscopy data (discussed below). The operational nature of the DFAA versus DCAA definitions means that amino acids liberated from difficult matrixes (e.g., humic substances) also could make up a part of DCAA. Total hydrolysable amino acid (THAA) is another term commonly used to denote both pools together, when the sample is hydrolyzed but DFAA are not independently determined. Because the DFAA pool is typically much smaller than DCAA, THAA values are often assumed to be similar to DCAA. 

A correlation between content of hydrophobic amino acids and surface activity of five different food proteins partially hydrolyzed with 0.1% pepsin has been reported (58), but exceptions were noted. Protein hydrolysates exhibiting large surface absorption were correlated with large foam stability and a large external hydrophobic region. It was concluded that protein hydrolysates with large surface hydrophobic regions adsorbed more readily at interfaces and rates of surface desorption were lower. However, secondary structures, as measured by optical rotatory dispersion and infrared spectra, and the content of the total hydrophobic amino acids in the protein hydrolysates showed no correlation with their foam stabilities (58). 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

Utilization of C-protein hydrolysate (cpm) Total cpm, C-amino acids... 

Unfortunately, there are numerous ways to circumvent the analysis of total amino acids, such as by the addition of ammonium salts, inexpensive amino acids, peptides and protein hydrolysates. Several approaches have been made to verify the authenticity of the total amino acid values. Rockland and Underwood (10) developed a paper chromatographic technique for quantitatively estimating the individual amino acids. 

For example, the removal of water from protein hydrolysates was carried out [11] by two methods, vacuum evaporation and lyophilization. (i) In vacuum evaporation, an aqueous aliquot of the protein hydrolysate, containing 5—25 mg of total amino acids, was transferred into a 125-ml flat-bottomed boiling flask with a PTFE-coated magnetic stirring bar. The sample flask was placed on a rotary vacuum evaporator and immersed in a water-bath at 60—70°C. Then the water was removed by slowly lowering the pressure (to prevent bumping) until the minimum pressure was attained, (ii) In lyophilization of the sample, an aliquot was placed in a 125-ml flat-bottomed flask as above and shell-frozen prior to being placed on an efficient lyophilizer to remove the water. 

The extent of hydrolysis of protein hydrolysates is measured by the ratio of the amount of amino nitrogen to the total amount of nitrogen present in the raw material (AN/ TN ratio). Highly hydrolyzed materials have AN/TN ratios of 0.50 to 0.60. To obtain the desired level of hydrolysis in a protein, a combination of proteases is selected. Serine protease prepared from Bacillus lichenifor-mis has broad specificity and some preference for terminal hydrophobic amino acids. Peptides containing terminal hydrophobic amino acids cause bitterness. Usually a mixture of different proteases is employed. The hydrolysis reaction is terminated by adjust-... 

The simple comparison of amino acid distributions in bacteria and phytoplankton reveals no obvious differences between these two possible autochthonous sources of dissolved proteins. Accordingly, the amino acid distribution in HMWDOM is similar to the distribution in phytoplankton, bacteria and sediment traps. The relationship between the amino acid distribution in total DOM and various particulate fractions is more variable. In general, it appears that amino acid distribution alone cannot identify the dominant source of dissolved proteins/peptides. However, assuming that the primary source of dissolved proteins in the ocean is bacteria and phytoplankton, these data support the interpretation that hydrolysable proteins in DOM and HMWDOM maintain a source-like amino acid distribution. 

It is a proteolytic enzyme, present in the intestine in its inactive form (zymogen), trypsinogen. Trypsinogen is converted into its active form, trypsin, by enteropeptidase, a specialized proteolytic enzyme secreted by intestinal cells. Some free trypsin formed also catalyses the conversion of trypsinogen into trypsin. Trypsin can also convert chymotrypsinogen and procarboxypeptidase into chymotrypsin and carboxypeptidase, respectively. Trypsin has different amino acid specificity when compared with other proteolytic enzymes. Trypsin hydrolyses those peptide bonds whose carboxyl groups are contributed by Lys or Arg residues and if the next residue is not proline. The number of smaller peptides resulting from trypsin action is equal to the total number of Arg and Lys residues in the protein plus one. 

If appropriate precautions have been taken in the preparation of a protein, and if oxygen is completely removed before hydrolysis, methionine will usually be recovered from acid hydrolysates in yields greater than 95 %. However, in some proteins (particularly those that are chemically modified) and in many peptides the methionine may be at least partially oxidized to the sulfoxide or sulfone forms, and even though these may be analyzed with amino acid analyzers (see below), the total yield of methionine (and oxidized products) is usually somewhat low. A good check on total methionine content in a peptide or protein is obtained by analyzing for methionine sulfone after performic acid oxidation, since methionine and its sulfoxides are quantitatively converted to the sulfone by this procedure. 

Protein Hydrolysate. Protein hydroly.sate is a solution of amino acids and short-chain oligopeptides that represeni the approximate nutritive equivalent of the ca.sein. lactaibu min. plasma, fibrin, or other. suitable protein from which il is derived by acid, enzymatic, or other hydrolytie method It may be modified by partial removal, and restoration or addition of one or more amino acids. It may contain dextrou or another carbohydrate suitable for intravenous infusion Not less than 50% of the total nitrogen present Ls in the form of o-amino nitrogen. It is a yellowish to rcd-ambet tran.sparent liquid with a pH of 4 to 7. 

Unfortunately, the 3-phenyl-2-thiohydantoins formed in the Edman stepwise degradation suffer racemisation (Davies and Mohammed, 1984) so that the method cannot be used to determine the configuration of amino acids in a peptide. This is not usually a serious limitation, but enantiomerisation is a perpetual hazard in peptide synthesis (Chapter 7). It is therefore desirable to determine if enantiomerisation has occurred at any residue. Such information could be important, for example, in dating bone proteins obtained in archaeological excavations. Examination of the chiral purity of the amino acids in a total acid hydrolysate is not satisfactory. 

Amino Acids. Kemp and Mudrochova (1973) determined amino acids and amino sugars by ion-exchange chromatography in 6N HCl hydrolysates of humic and fulvic acids from Lake Ontario sediments. They obtained total amino acids of 21.5% for humic acid and 12.6% for fulvic acid. Total amino sugars accounted for only 1.9 and 1.3% for humic acid and fulvic acid, respectively. They found the amino acid distribution in the humic acid resembled that of zooplankton and suspended sediment samples, with the exception of glycine which was higher in the sediments. This lends support for the assumed autochthonous nature of lake sediment organic matter. On the other hand, basic amino acid concentrations were low in the fulvic acid and its amino acid distribution resembled the combined form in the interstitial waters. 

Gas chromatography was used to analyze amino acids in 6N HCl hydrolysates of fulvic acid, humic acid, and humin from lake sediments (Lakes Suwa, Nakanuma, Yunoko, Haruna, Shoji, Motosu, and Biwa) (Yamamoto, 1983). Table 8 gives an example of analytical results of amino acids (Lake Haruna). The total amino acids for the seven-lake sediments accounted for 3-16% of humin, 11-21% of humic acid, and 4-24% of fulvic acid. The percentage of amino nitrogen in the total nitrogen in each fraction was 20-44% for humin, 21-36% for humic acid, and 4-30% for fulvic acid. 

The molar ratios of the amino-acid residues were determined after erythrocuprein was subjected to acid hydrolysis at 110°C for 20 to 96 hours. The method of Moore and Stein was used throughout (88, 89). The sulfhydryl content of human erythrocuprein was determined using the spectrophotometric assay of Boyer (90). Total half-cystine was analysed as S-carboxy methyl cysteine in acid hydrolysates (91) of reduced and alkylated erythrocuprein, or as cysteic acid in acid hydrolysates oxidized with performic acid. The data are summarized in Table 2. 

The total amino acid composition is determined by a complete hydrolysis of the peptide with 6 M HC1 at a high temperature. The hydrolysate is run on a paper electrophoresis to separate the amino acids. The different amino acids are visualized by staining with a dye called dinitrophenol. All amino acids produce yellow spots except proline, which appears blue. The intensity of color determines the amount of each amino acid in a particular spot. The different amino acid spots are identified by comparing their mobility with the known amino acids used as standard during electrophoresis. 

The circulins—As early as 1949, Peterson and Reineke characterized circulin as its sulphate. Total hydrolysis yielded D-leucine, L-threonine and L-K,y-diaminobutyric acid together with an optically active isomer of pelargonic acid. The existence of two components, found by Peterson and Reineke was later confirmed by the chromatographic separation of crude circulin into two major components, named circulin A and circulin B. In addition there was evidence for at least three other ninhydrin-positive, biologically active entities. In the hydrolysate of circulin A, L-isoleucine was found besides the amino acids previously reported . Quantitative amino acid analysis showed circulin A and B to be composed of L-a,y-diamino-butyric acid, L-threonine, D-leucine, L-isoleucine and ( + )-6-methyloctanoic acid in the molar ratio 6 2 1 1 1. After partial acid hydrolysis, fractionation and structure determination of the resulting peptides, circulin A and circulin B were formulated as cyclodecapeptides . Very recently, however, Japanese workers have revised the structure of circulin A. According to them, circulin A differs from colistin A only by a replacement of L-leucine in the latter by L-isoleucine Figure 1.7). 

Figure 1 Methionine contents 9 added to the reaction mixtures and covalently bound in the EPM products. H The methionine contents in the different reaction mixtures are as follows (1) 0.15 g Met/lg hydrolysate (2) 0.34 g Met/lg hydrolysate (3) 0.48 g Met/lg hydrolysate (4) 0.63 g Met/lg hydrolysate (5) 0.92 g Met/lg hydrolysate. Content of the covalently incorporated methionine in the EPM products. The methionine contents (percentage of the total amino acid content) were determined by amino acid analysis. A, Met concentration in the reaction mixtures (g/100 g) B, Met enrichment (%).

Radiation Effects on Solutions of Polymyxin. The decrease in the content of polymyxin and its biological activity, as well as the results of analyses for free and amide-like ammonia, total content of carbonyl substances, and the content of individual amino acids in the hydrolysates of irradiated samples are summarized in Table I. 

Polarimetry Total amino acids in proteins or hydrolysates, or pure amino acids Cumbersome separation of each amino acid from protein hydrolysates 15-19... 

Figure 5. Uptake of amino acids from pronase hydrolysate of untreated zein by perfused jejunal segments. Results represent the portion of total amino acids taken up and are expressed as the percent of the difference between the original and final amino acid concentrations in acid hydrolysates of the perfusate (AA - AAf) divided by the original concentration - AAf)l(AAJ] x 100.

The Committee also estimated exposure to carrageenan of infants of 12 months of age, based on a survey in France showing that consumption of formula represents 13.7% of total caloric intake at this age. Mean exposures were 6 mg/kg bw per day for milk- and soy-based formulas (0.03% carrageenan) and 22 mg/kg bw per day for hydrolysed protein- and/or amino acid-based liquid infant formulas (0.1% carrageenan). 

Figure 6 GLC analysis of soy bean meal hydrolysate. Experimental conditions 25 mg of soy bean meal hydrolyzed for 22 h at 110°C cleaned by cation exchange approximately 18pg total amino acid injected. (Reprinted with permission from Zumwalt RW, Kuo KCT, and Gehrke CW (eds.) (1987) Amino Acid Analysis by Gas Chromatography, vol. 1, p. 38. Boca Raton, FL CRC Press CRC Press, Boca Raton, Florida.)...

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