Lysine ninhydrin reaction

N-a-acety1-L-lysine (0.5 mmole) and glucose (1.0 mmole) are dissolved in 1 ml 0.1 M K2HP0 buffer solution. The solution is heated in a closed vessel for 5 h at 100°C. Then the solution is made 6 N with HC1 and heated at 105° for 24 h. The hydrolysate is evaporated in vacuo. A solution of the residue in 20 ml water can be used for the TLC production of furosine spots. For quantitative evaluation the same absorbance was assumed for the ninhydrin reaction product both arginine and furosine. 

Van Slyke s nitroua acid procedure (24), by the fonnol titmtion (25), or by use of the ninhydrin reaction (Sffi). These three methods do not always give the same value. The nitrous acid method probably gives the best value for the total amino nitrogen. The o-amino group may be expected to be titrated between pH 7.0 and 9.0, whereas that of the -amino nitrogen of lysine is nearer pH 8.6>11.5. 

Benoiton et al. (1971) have found that lysine and s-N-monomethyl-lysine have similar 570/440 nm ratios after reaction with ninhydrin as measured with the analyzer, and that e-N-dimethyllysine and e-N-trimethyllysine also have similar 570/440 nm ratios, but the 570/440 nm ratios of the two pairs are different. This provided a method for estimating from the 570/440 nm ratio the content of e-N-monomethyl-lysine and e-N-dimethyllysine in hydrolysates that are analyzed in systems in which these two derivatives are not separated. For other analyzer systems that have been used to study methylated lysines, see the references cited above and also Deibler and Martenson (1973). 

For example, if the modification reaction takes place via the lysine groups, the free lysine groups can be determined by ninhydrin [32,34], trinitrobenzenesulphonic acid [35,37], ortho pthalaldehyde [38,62], etc. 

Squibb [154, 155] has separated the free amino acids in bird liver extracts, using silica gel G layers on plastic sheets. The coloured reaction products with ninhydrin were evaluated photometrically at 525 nm, directly on the layer. Lysine, histidine, arginine, asparagine, alanine, valine and leucine were determined in this way. Recoveries lay between 90 and 106%. 

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