Total hydrolyzable amino acids

Henrichs, S.M., J.W. Farrington, and C. Lee. 1984. Peru upwelling region sediments near 15°S - II. Dissolved free and total hydrolyzable amino acids. Limnology and Oceanography 29 20-34. 

Note Humic substances (HS) are operationally defined as DOC that is retained on XAD-2 or XAD-8 resins. UDOM is operationally defined as DOC that is retained by a membrane with a 1 nm pore size and 1000 Da molecular weight cutoff. 13C-NMR spectroscopy was used for analysis of carbon functional groups, which are presented as a percentage of the total organic carbon in the sample. THNS, total hydrolyzable neutral sugars THAA, total hydrolyzable amino acids. 

TABLE II The Percentages of Bioavailable DOC (% BDOC) Accounted for as Total Hydrolyzable Neutral Sugars (THNS) and Total Hydrolyzable Amino Acids (THAA) in a Lake, Creek, and Marine Environment... 

Figure 9.34 Total hydrolyzable amino acid (THAA) concentrations (mg L-1) during (a) oxic and (b) anoxic decay of the dinoflagellate P minimum over different time periods. Hydroxylamino acids not shown, and nonprotein amino acids = /3-alanine and y-aminobutync acid. (Modified from Nguyen and Harvey, 1997.)...

Figure 15.17 Ratio of basic amino acids (B), (lysine and arginine) to the sum of basic plus acidic amino acids, (B+A), (aspartic and glutamic acids) versus carbon-normalized yields of total hydrolyzable amino acids (THAA). Symbols are as in Fig. 15.13.

Henrichs, S. M., Farrington, J. W., and Lee, C. (1984). Peru upweUing sediments near 15S. Part 2. Dissolved free and total hydrolyzable amino acids. Limnol. Oceanogr. 29, 20—34. 

Thurman (1985) provided a comprehensive assessment of the distribution of identifiable organic compounds in natural waters. Total hydrolyzable amino acids accounted for 2-3% of DOC in rivers and 3 -13 % of DOC in eutrophic lakes. Total hydrolyzable sugars accounted for 5-10% of DOC in rivers and 8-12% of DOC in lakes. Thurman (1985) also summarized the... 

The analysis of amino acids can be conducted on unmodified samples to quantify free amino acids, or total hydrolyzable amino acids can be quantified by conducting the analysis on samples that have been hydrolyzed for 24 h in 6 M HCl at 100 °C. In either case, amino acids must be derivatized prior to quantitative analysis. Derivatization is used to render amino acids less... 

Total hydrolyzed amino acids (THAAs) and total free amino acids (TFAAs). 

The major constituents of biomass are present at low levels in DOM. The median freshwater contains 1.3p,molL of total hydrolyzable amino acids, the most abundant of which are glycine (20%), aspartic acid (12%), alanine (10%), glutamic acid (9%), and serine (9%). Collectively, the amino acids account for 1.8% of the organic carbon in DOM and —18% of its nitrogen. Free amino acids also exist in freshwaters, but the median concentration of 0.3 p,mol is a factor of 4-5 lower than the concentration of bound amino acids. Elevated levels of serine and ornithine in the data set for free amino acids strongly suggest that some samples may have been contaminated by human fingerprints. 

Yields (%) are calculated based on input N2 or CO2 (for HCHO) from 100 mm CO2, 100 mm N2 and 100 mL H2O with or without 2 mmoles CaCCb in a 3.1 L flask at 23 C. Also included is an experiment with 10 mm of O2 added to the gas mixture. The results with a reducing atmosphere (% yields based on starting carbon in the form of methane) are given for comparison [taken from (3, 4). Yields of THAA (Total Hydrolyzable Amino Acids labeled in plate 2) are shown after hydrolysis in the absence (-) or presence (+) of ascorbate. The controls are not sparked and represent blanks. ND = not determined. 

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). 

Fig. 8. Batch culture of axenic Skeletonema costatum. a Numbers of living (LSc) and dead (DSc) cells, and concentration of soluble P (reactive) (SPR). b Concentration of particulate organic C (POC). c Concentration of dissolved free (DFAA) and dissolved total (DTAA) amino acids (free -I- hydrolyzed), and of dissolved monosaccharides (MCHO) and dissolved total carbohydrates (TCHO) [54]...

The use of amino acid analysis to characterize protein hydrolyzates is well known, and IEC is an integral part of the commercial amino acid analyzer.61 Determinabon of individual amino acids in physiological fluids such as plasma and cerebrospinal fluid can be important in the etiology of various diseases.62 63 Of particular current interest is the measurement of total... 

As another criterion of purity, the amino acid content of heparins should be determined. This is usually performed by ion-exchange88 or liquid89 chromatographic analysis of hydrolyzates. Reasonably pure heparin preparations contain < 1% of total amino acids, mostly L-serine and glycine. Heparin preparations should also be analyzed for residual solvents, and analytical (as well as biological) data be expressed on a dry basis. (Heparins equilibrated with atmospheric humidity contain up to 15%, or even more, of water.) Unless volatile materials are completely removed or accounted for, elemental analyses of heparin are meaningless. 

Considerable amounts of glutamic acid, glycine, and alanine, as well as smaller quantities of aspartic acid, serine, threonine, basic amino acids, leucine, phenylalanine, and cystine have been demonstrated in a total hydrolyzate of the nondiffusible fraction by Boulanger et al. (BIO). Using Deacidite resin, they separated this material into two polypeptide fractions, acid and alkaline, and found that glutamic acid, aspartic acid, leucine, and certain cystine derivatives were the chief constituents of the former, whereas the latter contained considerable amounts of glycine, basic amino acids, and alanine. 

Unidentified hydrolyzable N (UH-N) =% total hydrolyzable N - (% amino acid-N + NH3-N + amino sugar-N)... 

Lactic streptococci initiate casein degradation through the action of cell wall-associated and cell membrane-associated proteinases and peptidases. Small peptides are taken into the cell and hydrolyzed to their constituent amino acids by intracellular peptidases (Law and Sharpe 1978). Peptides containing four to seven residues can be transported into the cell by S. cremoris (Law et al. 1976B). S. lactis and S. cremoris have surface-bound peptidases and thus are not totally dependent on peptide uptake for protein use (Law 1979B). Some surface peptidases of S. cremoris are located in the cell membrane, whereas others are located at the cell wall-cell membrane interface (Exterkate 1984). Lactic streptococci have at least six different aminopeptidase activities, and can be divided into three groups based on their aminopeptidase profiles (Kaminogawa et al 1984). 

The nutritional values of foodstuffs are better correlated with the overall amino acid profile (compared to free amino acid composition). Not surprisingly, then, total amino-acid profile is much more commonplace in the food industry than is the analysis for free amino acids. In order to determine the amino acid composition of a foodstuff, proteins must first be hydrolyzed into their constituent free amino acids. 

PITC (phenylisothiocyanate) Aabs = 254 nm. Phenylthiocarbamyl amino acid derivatives are moderately stable at room temperature (1 day). PITC reacts well with both primary and secondary amino acids. Reaction time is approximately 5 minutes at room temperature. Excess reagent must subsequently be removed under vacuum. Also, for hydrolyzed samples, hydrochloric acid must be completely removed prior to derivatization. As a result, even though the actual reaction time is reasonably fast, the total time for various sample manipulations can add up to 2 hours. This is partially compensated by the extremely fast separation possible (12 minutes). Detection is by UV absorption only. Detection limits are typically in the high picomole range. Short column life can result due to unreacted PITC getting into the column. Unlike some of the other reagents, PITC quantifies tyrosine and histidine very well. PITC analysis is available as a commercially prepackaged system dubbed Pico-Tag by Waters Corporation. Representative references include 184-188. See Fig. 11 for a typical separation. 

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