FDNB-lysine

Table I shows a series of simple linear regression equations calculated from the data plotted in Figure 1. For the samples heated alone total lysine correlated ( r =0.55 ) badly with FDNB lysine and overestimated it at all levels of heating.

One would however expect this overestimation to be more profound since isopeptides are acid-labile and hence all lysine in this form is measured by total amino acid analysis. Also in the Maillard samples total lysine values overestimated FDNB lysine. 

Attempts to determine absolute values for the DAN method were complicated mainly by the problem that fluorescence intensity was found to increase with decreasing particle size ( even below 80 jim). For materials of the same particle size, as in the present study, the results correlated ( r = 0.85 ) quite well with the FDNB lysine and showed good reproducibility, attributable to the unique design of the fluorometer used ( 12 ). 

The dye-binding method with Acid Orange 12 was very similar in results to succinic anhydride, showing regression equations between ( near to ) SA and DAN. The DB method also underestimated FDNB lysine in the severely heat-damaged samples. 

Comparison with in vivo procedures Although the FDNB procedure proved to be a suitable reference method, there is no doubt that all methods should be ultimately compared to in vivo procedures. For this reason selected samples were also analyzed by plasma amino acid and digestibility methods. Preliminary results ( Table II ) show that plasma lysine results correlated very well with results for lysine digestibility and FDNB lysine ( r =0.95 ), reasonably well with those for dansyl chloride lysine, succinic anhydride reactive lysine and dye binding lysine, but poorly with total lysine. Although the absolute values were in many cases very different, it is apparent that all methods except total lysine can be used to at least indicate the extent of lysine damage. 

Lysine is an essential amino acid with an e-amino group on the side chain that can react with various food components. As known, reaction of the e-amine can render lysine nutritionally unavailable reducing the nutritional value of food. While the determination of total lysine is straightforward (it is stable to acid hydrolysis), the determination of available lysine is difficult as lysine adducts are labile to the standard acid hydrolysis. A solution to this problem consists of derivatizing the e-amino group with a chromophore such as l-fluoro-2,4-dinitrobenzene (FDNB) to form a derivate which is stable to optimized hydrolysis conditions [222]. 

Traditionally, most chemical methods for the direct determination of available lysine depended on the reaction of the e-amine with a chromophoric derivatizing reagent and then spec-trophotometric measurement. The most commonly employed chromophore is 1-fluoro-2,4-dinitrobenzene (FDNB), which was originally employed by Carpenter (102). Since then, numerous articles (103-106) have reported the direct (or indirect by difference) HPLC measurement of the FDNB-derivatized lysine. However, these methods can suffer from the problem that the reaction product of the FDNB-derivatized lysine (dinitrophenyllysine) is not entirely stable during acid hydrolysis (especially in the presence of carbohydrates) and correction factors are recommended (107). 

VH Booth. Problems in the determination of the FDNB-available lysine. J Sci Food Agric 22 658-664, 1971. 

You have a peptide that is a potent inhibitor of nerve conduction and you wish to obtain its primary sequence. Amino acid analysis reveals the composition to be Ala(5) Lys Phe. Reaction of the intact peptide with FDNB releases free DNP-alanine on acid hydrolysis. e-DNP-lysine (but not a-DNP-lysine) is also found. Tryptic digestion gives a tripeptide (composition Lys, Ala (2)) and a tetrapeptide (composition Ala(3), Phe). Chymotryptic digestion of the intact peptide releases a hexapeptide and free alanine. Derive the peptide sequence. 

From a rare fungus you have isolated an octapeptide that prevents baldness, and you wish to determine the peptide sequence. The amino acid composition is Lys(2), Asp, Tyr, Phe, Gly, Ser, Ala. Reaction of the intact peptide with FDNB yields DNP-alanine plus 2 moles of e-DNP-lysine on acid hydrolysis. Cleavage with trypsin yields peptides the compositions of which are (Lys, Ala, Ser) and (Gly, Phe, Lys), plus a dipeptide. Reaction with chymotrypsin releases free aspartic acid, a tetrapeptide with the composition (Lys, Ser, Phe, Ala), and a tripeptide the composition of which, following acid hydrolysis, is (Gly, Lys, Tyr). What is the sequence ... 

Several early workers reported the availability of lysine in breads (4, J 0, 21 ). Palamidis and Markakis (12) reported that the total lysine content decreased with baking or toasting. Available lysine contents significantly decreased as the heat exposure increased. This correlates highly with PER. However, existing techniques, such as fluorodinitrobenzene (FDNB) (2J ) and trinitrobenzenesulfonic acid (TNBS) 22), for chemically determining available lysine suffer interferences and inaccuracies. 

Fluorodinitrobenzene ( FDNB )-reactive lysine The direct FDNB method of Carpenter ( 8 ) was used with some further ( % J 0 ) modifications. 

In Figure 1 it is clear that all five procedures showed at least some measure of sensitivity to lysine damage in all heated samples and that generally with increased severity of heat treatment a corresponding decrease in reactive lysine was detected. The SA and DAN methods and to a smaller degree the DB method were more sensitive than the FDNB method, particularly for the most severely damaged samples. The total lysine method was least sensitive. 

Although other newer chemical methods are starting to take over as a method of choice for reactive lysine ( JJ9 ), in the present work the classical direct FDNB method was selected as standard reference since many times it has been shown to give results that correspond closely with those from both biological evaluation and in vitro enzymic digestion ( 19, 20 ). 

The SA method correlated quite well with the FDNB method, showing a greater sensitivity than total lysine but giving absolute values that were in some cases very different from that of FDNB. 

In the lower range the SA values were below FDNB values but above about 4 g lysine/16 g N, the SA levels were slightly higher. The difference may arise from the fact that FDNB tends to react with the E -Nl -linkage of blocked lysine ( 22 ). There may be also differences due to the failure of the various reagents to fully penetrate the high crosslinked material. The SA method may therefore better reflect the influence of poor digestibility on lysine availability. 

Table I. Simple linear regression equations correlation between FDNB-reactive lysine and lysine determined by the other methods...

If the four reactive-lysine methods studied are compared on the basis of simplicity and rapidity, the FDNB method is not preferred since it is fairly complicated, takes ca. 20 h per assay, and reqires special precautions due to the vesicant effects of the... 

The compound l-fluoro-2,4-dinitrobenzene (FDNB) reacts with free amino, imidazole, and phenolic groups at neutral to alkaline pH to yield the corresponding, colored dinitrophenyl (DNP) compounds. Thus, FDNB will react with the free, unprotonated a-amino groups on amino acids, as well as with the side chains of lysine, histidine, and tyrosine (Fig. 6-1). Dansyl chloride is another compound that is known to react with the unprotonated, N-terminal amino groups of peptides. De-rivatization of peptides with this compound yields fluorescent products that provide a very sensitive method of detection of the amino acid derivatives (Fig. 6-2). 

Besides reacting with the a-amino groups of proteins, FDNB also reacts with the -amino groups of the lysine residues, and an estimation of the e-DNP-lysine in the hydrolysate indicates how many of these amino groups are free in the intact protein. For all proteins studied reasonable... 

FDNB forms DNP derivatives with the side-chain amino groups of lysine residues and, to a lesser extent, the side-chains of histidine, cysteine, tyrosine, serine, and threonine residues. 

The gross protein value is probably the most commonly used biological method for evaluating proteins. Gross protein values measure the ability of proteins to supplement diets consisting largely of cereals and they correlate well with FDNB-reactive lysine figures. 

Table 13.6 Mean amounts of total lysine, FDNB-reactive lysine and truly digestible lysine in heat-treated meat and bone meals...

The increased use of synthetic lysine in foods presents a further problem. This amino acid has both amino groups available for reaction with FDNB. The resulting compound is soluble in ether and is not estimated by the Carpenter method. 

The chemical and enzymatic browning reactions of plant polyphenols and their effects on amino acids and proteins are reviewed. A model system of casein and oxidizing caffeic acid has been studied in more detail. The effects of pH, time, caffeic acid level and the presence or not of tyrosinase on the decrease of FDNB-reactive lysine are described. The chemical loss of lysine, methionine and tryptophan and the change in the bioavailability of these amino acids to rats has been evaluated in two systems pH 7.0 with tyrosinase and pH 10.0 without tyrosinase. At pH 10.0, reactive lysine was more reduced. At pH 7.0 plus tyrosinase methionine was more extensively oxidized to its sulphoxide. Tryptophan was not chemically reduced under either condition. At pH 10.0 there was a decrease in the protein digestibility which was responsible for a corresponding reduction in tryptophan availability and partly responsible for lower methionine availability. Metabolic transit of casein labelled with tritiated lysine treated under the same conditions indicated that the lower lysine availability in rats was due to a lower digestibility of the lysine-caffeoquinone complexes. 

The influence of pH, time and temperature of treatment was investigated only in relation to the loss of fluorodinitroben-zene FDNB-reactive lysine. Fig. 2 shows the influence of pH in solutions containing 5 % casein and 0.2 % caffeic acid stirred for 3 h at room temperature. In the presence of tyrosinase, there was an extremely narrow region for maximum reactive lysine loss at pH 7. At pH 6.8 and pH 7.5, the loss was already half that at pH 7. In the absence of tyrosinase, there was little loss of reactive lysine up until pH 8.8 however, at pH 10.0 only 73 % of the original reactive lysine remained. The loss only occurred in systems which were stirred and oxygenated. 

FDNB Methionine Methionine Tryptophan lysine sulphoxide... 

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