Non-enzymatic glycation induces structural modifications of myoglobin

Abstract

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25 degrees C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased alpha-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.

Fructose-induced modifications of myoglobin: Change of structure from met (Fe3+) to oxy (Fe2+) form

“…Although glycation of Mb has been reported from our laboratory [8,9], there has been no study on its fructation. Myoglobinuric renal failure in diabetic condition is known [37,38], and it may result from free radical damage involving 'free' iron release [39].…”

“…Findings from our laboratory indicate that HbA 1c exhibits structural and functional changes, and it may be a source of iron-mediated free radicals and oxidative stress in diabetic condition [4][5][6][7]. Glycation also induces modifications of another heme containing protein myoglobin [8,9], and is also associated with oxidative reactions. Glycated heme proteins may thus be associated with eliciting oxidative stress in diabetes mellitus, which is considered as a free radical mediated disease [10].…”

Structural stability of myoglobin and glycomyoglobin: a comparative molecular dynamics simulation study

“…However, several points should be taken into consideration in this regard: first of all, glycation is a process that includes the three stages of Schiff base formation, Amadori rearrangement, and final "AGE" (advanced glycation end products) generation, with the latest being irreversible cross-linked structures [24]. It is assumed that most of the experimental works that have been done on myoglobin concern the Amadori products [28], while the species that is modeled here would be closer to the first stage of glycation [29]. Thereafter, it has been reported that in the experimental setting, which is an average-time incubation (6 days) of the protein with glucose, release of iron occurs from the heme component [28], which may potentially lead to its destruction [30], and could be one cause of glycated myoglobin instability: apomyoglobin has lower stability than holomyoglobin [31].…”

“…It is assumed that most of the experimental works that have been done on myoglobin concern the Amadori products [28], while the species that is modeled here would be closer to the first stage of glycation [29]. Thereafter, it has been reported that in the experimental setting, which is an average-time incubation (6 days) of the protein with glucose, release of iron occurs from the heme component [28], which may potentially lead to its destruction [30], and could be one cause of glycated myoglobin instability: apomyoglobin has lower stability than holomyoglobin [31]. There is also the fact that the present work has been performed on human myoglobin, while the experimental works have been performed on either horse or whale myoglobins [27,28] and the study that reports a lack of glucose effect on increase of myoglobin amyloid formation (in contrast to ribose), has been made on a mutated species of whale apomyoglobin [27].…”

“…Thereafter, it has been reported that in the experimental setting, which is an average-time incubation (6 days) of the protein with glucose, release of iron occurs from the heme component [28], which may potentially lead to its destruction [30], and could be one cause of glycated myoglobin instability: apomyoglobin has lower stability than holomyoglobin [31]. There is also the fact that the present work has been performed on human myoglobin, while the experimental works have been performed on either horse or whale myoglobins [27,28] and the study that reports a lack of glucose effect on increase of myoglobin amyloid formation (in contrast to ribose), has been made on a mutated species of whale apomyoglobin [27]. Glycation effect on structure and activity of proteins would also depend on their incubation time with the carbohydrate molecule, as demonstrated in the case of aspartate aminotransferase whose activity amount differs upon brief or long incubating times with fructose [26].…”