Protein-bound Lysine Residues Research Articles

Quantification of the Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) in food

The Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) was recently identified as a new pyrrole amino acid resulting from the reaction of free and protein-bound lysine residues with the 1,2-dicarbonyl compound 3-deoxypentosone, pentose sugars, and degradation products of disaccharides. In the present work, simultaneous analysis of formyline and its structural analog, pyrraline, in enzymatically hydrolyzed food matrices was achieved on an RP-phenyl column by high-pressure liquid chromatography (HPLC) with UV detection. The presence of formyline was for the first time unequivocally verified in the hydrolyzates and standard samples by HPLC coupled to triple quad mass spectrometry due to identity of product ion spectra after targeted fragmentation of the molecular ion of formyline. A total of 68 food samples were analyzed. Low concentrations of formyline were detected in milk and whey products. In breakfast cereals, pasta, and bakery products, formyline was quantified in concentrations up to 34.8 mg/kg. In the outmost bread crust, 0.6 % of lysine residues can be modified to formyline. Moreover, it was shown by analysis of concentrated beer samples that both formyline and pyrraline are constituents of beer proteins. From our data, we conclude that at least 2–3 mg of formyline and 20–40 mg of pyrraline are ingested with the daily diet.

Preparation and analysis of the products of non-enzymatic protein glycosylation and their relationship to cross-linking of proteins

The presence of glycosylated protein-bound lysine residues has led to much speculation regarding changes in structure and function of the modified protein. The synthesis of hexose-lysine adducts and their separation using an amino acid analyser is described. These compounds are also produced during borohydride reduction and subsequent hydrolysis of modified proteins, and misidentification of these may occur depending upon precise chromatographic procedures. The possibility that glucose might participate in a cross-linking reaction between two protein-bound lysine residues was tested but no evidence for such a mechanism was found. The presence of 14C-labelled urinary hexosyllysine indicated that body protein breakdown in addition to ingested dietary hexosyllysine contributes to the excretion of this component.

Cystine-alkali reactions in relation to protein crosslinking.

The formation of lysinoalanine, lanthionine and β-aminoalanine in proteins, through the alkaline degradation of cystine residues to dehydroalanyl residues, is discussed. The formation of these amino-acids, during the dry heating of proteins, is also reported. It is shown that, during alkaline degradation of protein-bound cystine, amino or thiol-containing compounds present in the alkaline liquor can compete with protein-bound amino and thiol groups for addition to the dehydroalanyl residues formed as intermediates in the reaction. Results showing the relative importance of such competitive reactions are given. Evidence given indicates the one protein-bound cystine residue can, on alkaline degradation, yield two dehydroalanyl residues. The extent of addition of a series of alkylamines, to the transient dehydroalanyl residues, is shown to be dependent on the structure of the pendant alkyl group. Addition is governed by two factors, activity of the addendum, and the positioning of possible addenda in relation to the protein-bound dehydroalanyl residues, when the latter are formed. Dyeing experiments with alkylamine-treated wools show that the pendant alkyl groups influence dyeability. The results indicate that hydrophobic interactions in the modified protein have as great an effect on dyeability as do covalent crosslinks and ionic groups. Dye uptake is determined by the shape and size of the pendant alkyl groups introduced during the protein modification, rather then the enhanced basicity caused by introduction of the basic sidechains. The possibility of introducing hydrophobic sidechains into proteins, as amide derivatives of protein-bound lysine residues, by simple dry heating, is speculated upon, in relation to dry-heating of a fat-containing protein.KeywordsSilk FibroinWool FibreAlkaline DegradationWool KeratinCystine ResidueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.