Dissolved combined amino acids hydrolysis

Keil, R., and Kirchman, D. (1991b) Dissolved combined amino acids in marine waters as determined by vapor-phase hydrolysis method. Mar. Chem. 33, 243-259. 

J0rgensen, N. O. G., andjensen, R. E. (1997). Determination of dissolved combined amino acids using microwave-assisted hydrolysis and HPCL precolumn derivatization for labeling of primary and secondary amines. Mar. Chem. 57, 287-297. 

Robertson, K. Williams, P. Bada, J. Acid hydrolysis of dissolved combined amino acids in seawater a precautionary note. LimnoL Oceanog. 1987, 32, 996-997. 

Simply on the basis of the normal composition of marine organisms, we would expect proteins and peptides to be normal constituents of the dissolved organic carbon in seawater. While free amino acids might be expected as products of enzymic hydrolysis of proteins, the rapid uptake of these compounds by bacteria would lead us to expect that free amino acids would normally constitute a minor part of the dissolved organic pool. This is precisely what we do find the concentration of free amino acids seldom exceeds 150 xg/l in the open ocean. It would be expected that the concentration of combined amino acids would be many times as great. There have been relatively few measurements of proteins and peptides, and most of the measurements were obtained by measuring the free amino acids before and after a hydrolysis step. Representative methods of this type have been described [245-259]. Since these methods are basically free amino acid methods, they will be discussed next in conjunction with those methods. 

There are, unfortunately, no studies to date of the dissolved protein content of microlayer samples. With the recent development of many sensitive techniques for the analysis of amino-acid mixtures in seawater using liquid chromatography and fluorescence detectors (e.g., Dawson and Pritchard, 1978), it should be relatively simple to analyse for combined amino acids after hydrolysis of the microlayer samples. Analyses of free amino acids in the microlayer seem not to have been performed to date either, but since considerable degradation of surface-adsorbed proteins may take place as a result of UV irradiation, this may be a fruitful area for future research. 

Proline.—This is the only product of hydrolysis obtained from an ester fraction which is soluble in alcohol it is also much more easily soluble in water than the other products with which it is present and therefore is somewhat easily separated, as it remains in the mother-liquor after these have crystallised out. The solution, in which it is contained, is evaporated to dryness and extracted with absolute alcohol the combined alcoholic extracts from the several fractions are evaporated to dryness and taken up by absolute alcohol several times, so as to remove small amounts of the other amino acids, which, though insoluble in alcohol, are dissolved when proline is present. 

Protein Hydrolysates. Instead of ethyl hippurate, a peptic hydrolysate of ovalbumin was used as substrate for the resynthesis reaction (64). This substrate (300 mg) was dissolved in water, adjusted to pH 6.0 with NaOH and to 0.9 ml with additional water. An amino acid ester was added to produce a 22.2mM solution and the mixture preincubated at 37°C for 15 min. Papain (3 mg), dissolved in 0.1M L-cysteine (0.1 ml), was combined with the above-mentioned preincubation mixture and incubation carried out at 37°C. After 2 hr, 0.1N NaOH (10 ml) was added to stop the enzymatic reaction and the resulting solution allowed to stand for 3 hr to hydrolyze completely the remaining amino acid ester as well as the ester group from the peptide product. The free amino acid produced from the base-catalyzed hydrolysis of the amino acid ester was determined with an amino acid analyzer. The amount of the amino acid incorporated was obtained by subtracting the determined value from the initial concentration of amino acid ester. The data obtained with the same L-amino acid esters as used in the model experiment (above) are plotted along the ordinate of Figure 3. An excellent correlation is found between the data from the model experiment and those from this experiment using a protein hydrolysate. In Table III data are shown for the extent of covalent incorporation after 2 hr of various amino acid ethyl esters into the protein hydrolysate. There is a close relationship between... 

On the other hand, the esters of the polypeptides are of the greatest importance and they are prepared by the action of alcoholic hydrochloric acid. Hydrolysis of the polypeptide does not occur if prolonged heating be avoided, nor does hydrolysis occur when the esters are saponified by dilute cold caustic alkali. The esters have served in particular for the further synthesis of polypeptides and for the isolation of dipeptides from mixtures on treatment with alcoholic ammonia, the dipeptide esters are converted into their diketopiperazines. They are not soluble in petroleum ether and they are soluble with difficulty in ether, and they thus differ from amino acid esters. Chloroform dissolves them, and in this solvent their combination with acid chlorides has been generally effected. 

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