Free and Bound Amino Acids

Generally, amino acids, proteins, and their products are synthesized by enzymes according to current dogma (DNA makes mRNA makes protein). The occurrence of extracellular proteins involves the dynamics of cellulose and callose biosynthesis and cell wall development (9, 106). Against this background, the emergence of a pattern in the cell walls of woody plants may be studied (62). At the cellular levels, macromolecular biosynthesis is commonly believed to involve the dynamics of membrane transformation and flow (100, 76). 

Components of membrane transformation systems include the nucleus with its DNA, nonhistone chromosomal proteins, and the derived informational molecules (e.g., mRNA) for protein synthesis. The mRNA moves into the cytoplasm through the transformation of membranes. For example, the rough endoplasmic reticulum contains ribosomal RNA with its templates for protein synthesis. The system evolves with cytoplasmic organelles and substrates to influence structure and function. During this process the vacuoles, microtubules, endoplasmic reticulum, subcellular organelles, and plasmalemma may release specific metabolic nitrogenous products (conjugates, enzymes) internally or externally. Some of these may be involves in the biosynthesis of cutin and suberin (76). 

Free amino acids have been detected in alcohol extracts of wood of various species (52, 98). Bound amino acids in sapwood, heartwood, and reaction wood 

Tyrosine is the source of p-hydroxybenzaldehyde when milled wood lignin is oxidized with nitrobenzene. The loss of more than 20% of individual amino acids in the presence of milled wood lignin and a number of sugars and glucans was demonstrated. Numerous unidentified compounds were detected some may arise as reaction products among amino acids and sugars (e.g., 57). 

Extracellular free amino acids are usually those commonly found in protein. Some non-protein amino acids, such as y-aminobutyric acid and )8-amino acids, also occur. Low levels of )8-amino acids are usually products of nucleic acid degradation. 

---

G.G. Smith, R.C. Evans, The Effect of Structure and Conditions on the Rate of Racemization of Free and Bound Amino Acids, in Bio geochemistry of Amino Acids, edited by P.E. Hare, T.C. Hoering, J. King, John Wiley Sons, Ltd, New York, 257 282 (1980). 

Smith, G.G. and Evans, R.C. (1980). The effect of structure and conditions on the rate of racemisation of free and bound amino acids. In Biogeochemistry of Amino Acids, ed. Hare P.E., Hoering T.C., and King K. Jr, John Wiley, New York, pp. 257-282. 

More than 700 constituents have been identified in aroma extracts of roasted coffee. Heterocyclic aroma components represent the greatest amount of the steam volatile aroma complex (80 - 85 %) which amounts to 700 -900 ppm in medium roasted Arabica coffees. The concentration of individual components varies depending on coffee varieties and roasting conditions. Typical components are formed by thermal degradation of free and bound amino acid and chlorogenic acid precursors. Compared to other roasted foodstuffs, sulfur containing constituents and phenols are formed in high amounts and contribute to desirable coffee flavor or off-flavor. 

Scoccianti, V., Variations in the content of free and bound amino acids during dormancy and the first tuber cycle in Helianthus tuberosus tuber explants, Giom. Bot. Ital., 117, 237-245, 1983. 

A brief discussion of the chemical reactivity of the products of these enzymes is central to our proposed use of these enz)nnes as antinutritive bases of resistance. Polyphenol oxidase (PPO) and peroxidase (POD) oxidize phenolics to quinones, which are strong electrophiles that alkylate nucleophilic functional groups of protein, peptides, and amino acids (e.g., -SH, -NHof -HN-, and -OH)(Figure 1)(53,63-65). This alkylation renders the derivatized amino acids nutritionally inert, often reduces the digestibility of protein by tryptic and chymotryptic enzymes, and furthermore can lead to loss of nutritional value of protein via polymerization and subsequent denaturation and precipitation (63,66-69). POD is also capable of decarboxylating and deaminating free and bound amino acids to aldehydes (e.g., lysine, valine, phenylalanine. 

The natural occurrence and the role of free and bound amino acids, proteins, nucleic acids, and related products (e.g., 8, 42, 116) are described in relation to factors determining the composition of the lignocellulosic cell wall. The biosynthesis of extraneous nitrogenous compounds involves a sequence of events that stem from molecular reactions inside cells, on the surfaces of cells, and in the soluble component of plant cell walls. 

Takano, Y Kaneko, T Kobayashi, K. Hiroishi, D. Ikeda, H. Marumo, K. Experimental verification of photostability for free- and bound amino acids exposed to y-rays and UV irradiation. Earth Planets Space 2004, 56, 669-674. 

Himdin [8001-27-2] is a polypeptide of 66 amino acids found ia the saUvary gland secretions of the leech Himdo medicinalis (45). It is a potent inhibitor of thrombin and biads to y-thrombia with a dissociation constant of 0.8 x 10 ° M to 2.0 x lO " M. Himdin forms a stable noncovalent complex with free and bound thrombin completely iadependent of AT-III. This material has now been cloned and expressed ia yeast cells (46,47). Its antigenic poteatial ia humans remains to be estabUshed. 

Bettger, W.J. 1989. The effect of dietary zinc deficiency on erythrocyte-free and membrane-bound amino acids in the rat. Nutr. Res. 9 911-919. 

Stein et al. found in the course of experiments dealing with free and conjugated urinary amino acids in Wilson s disease (S9) that besides a marked aminoaciduria, almost a twofold increase in the excretion of all bound amino acids could be observed. As compared with normal urine (S8), unusual amounts of conjugated leucine, isoleucine, and valine are excreted in cases of Wilson s disease. Also the increase of glutamic acid, aspartic acid, and phenylalanine after urine hydrolysis is much more distinct in this disease than in normal conditions. Other bound amino acids are at or below normal levels. 

R Liardon, R lost. Racemization of free and protein-bound amino acids in strong mineral acid. Int J Pept Prot Res 18, 500, 1981. 

There are two major categories for amino acid analysis (a) free amino acid analysis and (b) determination of total amino acid content. The total amino acid content includes contributions from the free amino acids and the amino acids that are originally protein bound. These protein-bound amino acids must first be liberated before chromatographic analysis. This necessitates a more extensive, and problematic, sample preparation. Because the sample preparation procedures are so disparate, it is convenient to address these two categories of amino acid analyses separately. It should be noted that while the sample preparations for these analyses are quite different, both utilize essentially the same chromatographic techniques for the second stage of amino acid analysis. 

It may also be surprising how easily this racemization may occur. Friedman and Liardon (126) studied the racemization kinetics for various amino acid residues in alkali-treated soybean proteins. They report that the racemization of serine, when exposed to 0.1M NaOH at 75°C, is nearly complete after just 60 minutes. However, caution must be used when examining apparent racemization rates for protein-bound amino acids. Liardon et al. (127) have also reported that the classic acid hydrolysis, employed to liberate constituent amino acids, causes amino acids to racemize to various degrees. This will necessarily result in D-isomer determinations that are biased high. Widely applicable correction factors are not possible since the racemization behavior of free amino acids is different from that of amino acid residues in proteins (which can be further affected by sequence). Of course, this is not a problem for free amino acid isomer determinations since the acid hydrolysis is unnecessary. Liardon et al. also describe an isotopic labeling/mass spectrometric method for determining true racemization rates unbiased by the acid hydrolysis. For an extensive and excellent review of the nutritional implications of the racemization of amino acids in foods, the reader is directed to a review article written by Man and Bada (128). 

That summary is based on the reports of a well-conceived and carefully executed research program carried out by Rohan. Mohr et al. (7) extended these studies and was able to draw additional conclusions. First, without exception, free amino acids are much more sensitive to destruction in this system than the peptide-bound amino acids. Second, differences in the stability of amino acids under these conditions are not great —from 25% loss for isoleucine to 68.5% for lysine, over a relatively short period of time. In this system the reducing sugars must be the limiting factor, since the glucose and fructose are completely destroyed or removed. Third, neither cystine nor cysteine are reported to be present, and the only other sulfur-containing amino acid, methionine, is present at a much lower concentration than any other amino acid. Clearly, as we shall see later, cocoa would probably have a considerably different flavor if cysteine or cystine were present in the fermented beans. 

C.R. Lytle and E.M. Perdue, Free, proteinaceous, and humic-bound amino acids in river water containing high concentrations of aquatic humus, Environ. Sci. Tech. 15 (1981) 224-228. 

Some 350 other volatiles have been identified in chocolate aroma, and about 10% are pyrazines (48— 51) and quinoxalines (52). During roasting 49% of the total free amino acids are lost with only 4% of the bound amino acid being lost (53). 

The Maillard reaction commonly occurs in food products and during food processing. A typical or pure Maillard reaction is simply the reaction of a sugar and an amino acid. Strictly speaking, the sugar must be a reducing carbohydrate and the amino acid can be either free or bound, as a peptide or protein. The reaction generates not only volatile compounds, which provide odor, but also odorless nonvolatile compounds, some of which are colored. 

Incubations of crude pH 5 Supernatant with several C14-aminoacyl sRNA preparations, differing only in the nature of the C14-amino acid, showed that all amino acids tested were incorporated into ribosomal protein. With combined transferases I and n, the results presented in Table VII indicated that all of the amino acids tested were also incorporated in the presence of these purified fractions (8). When either of the transferases was omitted from these incubations, little amino acid transfer was observed with any of the C14-aminoacyl sRNA preparations. Variations in total amounts of C14 incorporated, as shown here, are probably due to variations in the specific radioactivity of the various sRNA-bound amino acids used. These purified transferase preparations did not catalyze the incorporation of free amino acids into sRNA or ribosomes. 

The details of the reaction mechanism with DCC were given in Chapter 43, p. 1172, and can be shown more easily if we mark the polymer and spacer as P and the cyclohexyl groups as CR The DCC is protonated by the free carboxylic acid and is then attacked by the carboxylate anion. The intermediate is rather like an anhydride with a C=NR group replacing one of the carbonyl groups. It is attacked by the amino group of the polymer-bound amino acid. The by-product is dicyclohexy-... 

---