Abstract
The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.
Highlights
Non-enzymatic modification of proteins through the Maillard reaction has recently gained new attention
This study provides further evidence that GlcN is a highly reactive monosaccharide, leading to heme reduction, displacement and significant modifications of protein conformation, involving fragmentation and formation of amyloidal-type fibrils
Production of α-dicarbonyl compounds (α-DC) and advanced glycation end-products (AGEs) are suggested to play a primary role in protein aggregation rapidly observed after 3 days of reaction
Summary
Non-enzymatic modification of proteins through the Maillard reaction has recently gained new attention. The endogenous reaction may form modified protein structures that affect cell metabolism, whereas the exogenous reaction occurs during food processing and confers positive qualities regarding flavour, colour, antimicrobial activity [9] and antioxidant capacity [10,11]. Beside these positive effects, there is a controversy on the harmful consequences that some of the Maillard reaction products, advanced glycation end-products (AGEs) in particular, exert on human health. Well known examples of these compounds are acrylamide and 5-hydroxymethylfurfural, referred as neo-formed contaminants [12]
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