Spectroscopic studies of the protein-methylglyoxal adduct.

Abstract

Spectroscopic measurements are reported for the effects of pH, time, solvent, and chemical modification of arginine and lysine side chains on the reaction of proteins with methylglyoxal. The reaction responsible for the appearance of a brown coloration and increased submolecular electronic activity in the proteins involves the e-amino groups of the lysine residues. It is concluded that the primary step in the reaction involves the formation of a Schiff base linkage between the lysine side chain and methylglyoxal. These findings reaffirm the concept that, by the formation of Schiff bases, aldehydes can act as electron acceptors in charge transfer interactions with proteins.Considerations (1, 2) of the possible biological role of aldehydes and ketones in acting as electron acceptors for proteins have led us to study the interactions of proteins with methylglyoxal. These studies have shown that when proteins such as casein, collagen, bovine serum albumin, and lysozyme react with methylglyoxal they assume a stable brown color and exhibit a greatly enhanced electronic conductivity and electron spin resonance activity compared with the normal (white) unreacted proteins (3-5).Scintillation counts by W. M. Arnold and J. Behi using 14C-labeled methylglyoxal (personal communication) and weight increase measurements have shown that after reaction with methylglyoxal for 5 days the resulting brown proteins contain on average 8-12% methylglyoxal on a dry weight basis. Amino acid analyses for the adducts of bovine serum albumin and of casein with methylglyoxal, performed by G. E. Siefring, Jr., at Northwestern University (personal communication) indicate that the arginine (;z100% reacted) and lysine residues (80-90% reacted) are the ones that react most strongly with methylglyoxal. We have blocked the arginine side chains of albumin with cyclohexanedione, using the procedure described by Smith (6), and this has produced no observable difference in the way the protein reacts with methylglyoxal to produce the brown color and electronic activity. However, when the E-amino groups of the lysine side chains of albumin and casein have been reductively dimethylated by the method of Lin et al. (7), upon reaction with methylglyoxal the proteins remain white and do not exhibit enhanced electronic activity. These results indicate that the reaction of particular importance in these studies involves the protein lysine residues, and the following spectroscopic studies were made as a step toward understanding the nature of this reaction. METHODSMeasurements were obtained by using a dual beam Beckman model 35 spectrophotometer and quartz sample cells (optical pathlength, 1 cm). For the spectra described here, 0.15 mM bovine serum albumin was allowed to interact with approxi- mately 10 mM methylglyoxal in water at 297 + 1 K; and the reference system consisted of an aqueous solution of methylglyoxal at the same concentration and pH as in the albumin/ methylglyoxal sample. The bovine serum albumin (fraction V) was obtained from Sigma. The...

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

“…In experiments where DJ-1 was added to the initial glycation mixtures, GAPDH activity was measured by adding 5 l of a 100-fold diluted glycation mixture to a cuvette containing 150 l of substrates. Glycation/deglycation of BSA lysines was followed by measuring the absorbance at 333 nm (31). BSA was incubated in buffer at 22°C in the absence or presence of 6 mM MGO and DJ-1, as indicated.…”

Free Radicals Generated during the Glycation Reaction of Amino Acids by Methylglyoxal

“…Previous investigations of the reactions of methylglyoxal with protein or methylamine also suggested that a condensation product, which was not well characterized, served as the electron donor and that methylglyoxal acted as an electron acceptor (41)(42)(43).…”

Oxidation-Reduction Properties of Methylglyoxal-modified Protein in Relation to Free Radical Generation

“…These species include N ⑀ -(carboxyethyl)lysine (22), imidazolone compounds (23), and imidazolium cross-link species, methylglyoxal-lysine dimer (24 -26). In addition to these AGEs, several investigations have also shown by electron paramagnetic resonance (EPR) spectroscopy that unidentified protein free radicals were produced during the reaction of methylglyoxal with proteins, such as bovine serum albumin (BSA) and casein (27,28). In our previous report (29), the free radical was assigned to be the radical cation of the cross-linked Schiff base on the basis of the detailed analysis of EPR spectra observed from the reaction mixture containing methylglyoxal and alanine.…”