The present study deals with the mechanistic reaction pathway of the α-dicarbonyl compound methylglyoxal with the guanidino group of arginine. Eight products were formed from the reaction of methylglyoxal with N(α)-tert-butoxycarbonyl (Boc)-arginine under physiological conditions (pH 7.4 and 37 °C). Isolation and purification of substances were achieved using cation-exchange chromatography and preparative high-performance liquid chromatography (HPLC). Structures were verified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. 2-Amino-5-(2-amino-4-hydro-4-methyl-5-imidazolinone-1-yl)pentanoic acid (3) was determined as the key intermediate precursor within the total reaction scheme. Kinetic studies identified N(δ)-(5-methyl-4-oxo-5-hydroimidazolinone-2-yl)-L-ornithine and N(7)-carboxyethylarginine as thermodynamically more stable products from compound 3. Further mechanistic investigations revealed an acidic hydrogen at C-8 of compound 3 to trigger aldol condensations. This reactivity of compound 3 allowed for the addition of another molecule of methylglyoxal to form products, such as N(δ)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-l-ornithine and argpyrimidine.

The present study investigated chemically modified gelatin biopolymer films. Gelatin solutions were treated with glyoxal and glycolaldehyde, respectively, at concentrations ranging from 0.25 to 7.5 wt % based on gelatin. From these solutions, films were produced under defined conditions and characterized with different chemical and physical methods. N(epsilon)-carboxymethyllysine (CML), glyoxal-derived lysine dimer (GOLD), and 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline (imidazolinone) were analyzed as chemical parameters for protein modification by reversed-phase high-performance liquid chromatography (RP-HPLC) and fluorescence detection after post-column o-phthaldialdehyde derivatization. An increase in the content of these substances with increasing concentrations of carbonyl modifiers correlated with the loss of available free lysine and arginine residues. Swelling, solubility, and mechanical properties (Young's modulus, stress and strain at break) showed the relationship with the degree of monovalent modification and cross-linking as well. The determination of unreacted glyoxal and glycolaldehyde suggested a different mechanism of cross-linking induced by glyoxal versus glycolaldehyde as reactive intermediates in Maillard chemistry.

Alpha A-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), αA-crystallin becomes a better chaperone. Alpha A-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of αA-crystallin by MGO (with 20-500 μM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of βL- and γ-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured βL- and γ-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of αA-crystallin to assist in the refolding of denatured MDH. However, MGO-modification of chaperone-null TNS-bound αA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of αA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the αA-crystallin responsible for the chaperone function and refolding are the same in the native αA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance αA-crystallin's ability to refold denatured proteins.

The present study deals with the characterization of the ripening of cheese. A traditional German acid curd cheese was ripened under defined conditions at elevated temperature, and protein and amino acid modifications were investigated. Degree of proteolysis and analysis of early [Amadori compound furosine (6)] and advanced [N(ε)-carboxymethyllysine (4), N(ε)-carboxyethyllysine (5)] Maillard reaction products confirmed the maturation to proceed from the rind to the core of the cheese. Whereas 6 was decreased, 4 and 5 increased over time. Deeper insight into the Maillard reaction during the ripening of cheese was achieved by the determination of selected α-dicarbonyl compounds. Especially methylglyoxal (2) showed a characteristic behavior during storage of the acid curd cheese. Decrease of this reactive structure was directly correlated to the formation of 5. To extend the results of experimental ripening to commercial cheeses, different aged Gouda types were investigated. Maturation times of the samples ranged from 6 to 8 weeks (young) to more than 1 year (aged). Again, increase of 5 and decrease of 2 were able to describe the ripening of this rennet coagulated cheese. Therefore, both chemical parameters are potent markers to characterize the degree of maturation, independent of coagulation.

The influence of different pretreated film forming solutions (FFSs) on the physical, chemical and barrier properties of gelatin films was studied. Hydrolyzation with hydrochloric acid and acetylation with acetic acid anhydride at different pHs were performed. Subsequently, gelatin was crosslinked with glyoxal to result in biopolymer films with properties predetermined by the rheological behavior of the respective FFS. Gel time, storage modulus and tan d of FFSs were obtained from small-strain oscillatory measurements and were correlated with swelling and solubility of the resulting films as well as with chemical analysis of lysine and arginine and the respective amino acid modifications N 3 -carboxymethyllysine (CML), glyoxal derived lysine dimer (GOLD) and 5-(2-imino-5-oxo-1imidazolidinyl)norvaline (imidazolinone). In general, chemical modification reduced the storage modulus whether it was hydrolyzation, acetylation or modification with glyoxal. Treatment by hydrolyzation or acetylation led to differences in gel time and in the ratio between viscous and elastic portion of the different hydrogels, also affecting the relationship between monovalent amino acid modification and bivalent crosslinking after glyoxal addition. Thus, the structural character of FFS hydrogels and of the protein network of films both determined the physical and barrier properties of chemically modified gelatin films.