Lysinoalanine, cross-links

Tolgyesi and Fang [68] have found that alkaline amine solutions react differently with human hair. With human hair, all amines examined, including pentyl amine, compete less effectively with the amino and mercaptan residues of the hair for the dehydroalanine intermediate. As a result, more lanthionine and lysinoalanine cross-links form than amine adduct, when human hair is the substrate. This is probably because diffusion rates are slower into human hair, decreasing the effective concentration of free amine in the fibers. Therefore, these species cannot compete as effectively for the dehydroalanine intermediate therefore, lanthionine and lysinoalanine are formed. 

V-1 from acid and alkaline hydrolyzates, SCX-HPLC of amino acids, a mixture of purified crosslinks and hydroxylysine b purified cross-link V-2 c amino acids from an acid hydrolyzate (6 M HCl) of reduced bovine dentin retained on a phenylboronate agarose column after purification as high molecular weight fractions by repeated size exclusion chromatography d as c, alkaline hydrolyzate (2 M KOH). Injections (c, d) resulted from 18 and 52 mg collagen originally hydrolyzed, respectively. 1 = 111 (HP) 2 = V-2 3 = IV 4 = V-1-1 (DHLNL) 5 = HLNL (bovine tendon) 6 = VI (histidinoalanine ) 7 = hydroxylysine 8 = VI (lysinoalanine). 

Other conversions to unnatural residues occur when most proteins are exposed to high pH (80, 81,82). The high pH causes a -elimination of a cystine (see Figure 16) or O-substituted serine or threonine, with the formation of a dehydroalanine or a dehydro-a-aminobutyrate. Such products are subject to nucleophilic attack by the e-amino group of a lysine to form a cross-linkage, such as lysinoalanine, or attack by cysteine to form lanthionine. Walsh et al. (81) have taken advantage of the formation of these cross-links to produce avian ovomucoids that have nonreducible cross-links and have lost the antiprotease activity of one of their two inhibitory sites (see Figure 17). 

Nutritional and Physiological Effects of Alkali-Treated Proteins. The first effect of the alkaline treatment of food proteins is a reduction in the nutritive value of the protein due to the decrease in (a) the availability of the essential amino acids chemically modified (cystine, lysine, isoleucine) and in (b) the digestibility of the protein because of the presence of cross-links (lysinoalanine, lanthionine, and ornithinoalanine) and of unnatural amino acids (ornithine, alloisoleucine, / -aminoalanine, and D-amino acids). The racemization reaction occurring during alkaline treatments has an effect on the nitrogen digestibility and the use of the amino acids involved. 

Alkali has long been used on proteins for such processes as the retting of wool and curing of collagen, but more recently it has received interest from the food industry. Alkali can cause many changes such as the hydrolysis of susceptible amide and peptide bonds, racemization of amino acids, splitting of disulfide bonds, beta elimination, and formation of cross-linked products such as lysinoalanine and lanthionine. 

Partial removal of the phosphate groups of phosvitin by -elimination in alkaline solution results in a decreased in vitro initial rate of hydrolysis by trypsin (268). The decreased rate of hydrolysis might be a result of (a) a change in conformation of the protein on removal of the phosphate groups, (b) cross-linking by the reaction of the dehydroalanine residues with lysine residues (to form lysinoalanine), or (c) racemization of some of the residues by the alkaline treatment. 

A test to discover whether wool has been damaged in processing is to determine the fraction of the wool that dissolves under standard conditions in a solution containing urea, as a hydrogen bond breaker, and a reducing agent, usually bisulfite. Add damage increases the solubility of treated, compared with untreated wool, because add hydrolyzes peptide bonds in the protein chains. Alkali treatment decreases the solubility, because redudble cystine residue disulfide cross-links are slowly replaced by nonreducible lanthionine and lysinoalanine crosslinks in alkali (see Section 5.3.4). 

Cystine residues in wool are attacked by alkali [13,242], and two new cross-links lanthionine and lysinoalanine—are formed. A mechanism for the reaction of cystine residues with alkali has been suggested. 

Treatment of wool with water or neutral buffer solution [13,242] at temperatures above 50°C causes the formation of lanthionine and lysinoalanine residue cross-links and, therefore, decreases the solubility of wool in urea-bisulfite solutions. When wool is heated in water in a sealed tube, it contracts at temperatures between 128 and 140°C, depending on the rate of heating. 

When wool is treated with alkali or hot water in the absence of added agents, cross-linking also occurs, to form lanthionine and lysinoalanine [253]. 

Dry heat in air causes less damage to wool than wet heat does [13,267,268]. Above 140°C, yellowing or scorching occurs lanthionine, lysinoalanine, and isopeptide cross-links are formed, and solubility in urea-bisulfite solution decreases. Cysteic acid residues are formed... 

More than 50 xenobiotic amino acids are known. Ammonia, which adds to dehydroprotein to form 2,3-diaminopropionic acid (P-aminoalanine, 2-41), was used in the prevention of lysinoalanine and other cross-link amino acids formation. 

In heat treated or stored food products several amino acids are not fully available because of derivatization or crosslinking reactions. Since 30 years furosine is known as a useful indicator of early Maillard reaction which is applied in food science, nutrition and medical biochemistry. Recently more sensitive analytical methods for furosine determination are available which have again increased the attractivity of this important indicator. Lately, N -carboxymethyllysine (CML) became available as another marker of special interest, because CML is a more useful indicator of the advanced heat damage by Maillard reaction than furosine. In addition, CML has the advantage to indicate reactions of lysine with ascorbic acid or ketoses such as fructose. Indicators for protein oxidation of sulfur amino acids are methionine sulfoxide and cysteic acid. An established marker for cross-linking reactions is lysinoalanine, which also indicates protein damages due to processing under alkaline conditions. Other markers formed as a consequence of alkaline treatment are D-amino acids. 

Racemization reactions also lead to protein cross-linking reactions. These reactions occur because of the formation of cross-linking compounds such as lysinoalanine (LAL). The formation of LAL not only renders the Lys that participates in the reaction unavailable to the animal, but it also decreases the digestibUity of total protein and other AA because LAL impairs the approach of proteolytic enzymes to the peptide chain (Boschin et al, 2003). However, because LAL formation occurs more readily at a higher pH, this reaction is more prevalent in alkali treated proteins compared with proteins simply exposed to heat treatments (Bunjapamai et al, 1982). Lysinoalanine content of a feed can be determined when analyzing feeds for AA content. 

As reported by Friedman (1982), the major factor controlling the production of lysinoalanine, once the dehydroalanine precursors are formed, could be the location and availability of "partners for crosslink formation. When they are adjacent or close by, lysinoalanine formation could be facilitated. When treatment allows it, more lysinoalanine could be formed, even involving cross-chain links. Thus, the capacity for forming LAL would vary not only with the treatment applied but with the nature of the protein, involving its secondary or tertiary structure as well as its primary one. 

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