Protein and Low Molecular Mass Thiols as Targets and Inhibitors of Glycation Reactions

Abstract

Protein glycation has been implicated in the aging process as well as the complications of diabetes (retinopathy, neuropathy, nephropathy, and atherosclerosis). The nitrogen substituents of Lys, Arg, and His residues and the N-terminus of proteins are known to be readily glycated. As the thiol group of Cys is a powerful nucleophile, we hypothesized that Cys residues should also be targets of glycation and that low molecular mass thiols may act as protective agents. In this study the role of thiol glycation, induced by dicarbonyls, in protein cross-link formation and damage prevention is examined. It is shown that incubation of creatine kinase with glyoxal results in protein cross-link formation, with this occurring concurrently with loss of thiol groups, enzyme inactivation, and formation of S-carboxymethylcysteine, a product of glyoxal adduction to Cys residues. Cross-links have also been detected between N-acetylcysteine and the Lys-rich protein histone H1, demonstrating the formation of thiol-glyoxal-amine cross-links. Mass spectrometry has been used to characterize some of these cross-links as 2-(alkylthio)acetamides. A range of low molecular mass thiols have been shown to inhibit dicarbonyl adduction to, and cross-linking of, the thiol-free protein lysozyme, consistent with these thiols being alternative (sacrificial) targets of glycation. Some of these thiols are more efficient modulators of glycation than established glycation inhibitors such as aminoguanidine. These data demonstrate that thiols are facile targets of glycation and that low molecular mass thiols are potent glycation inhibitors. These data may aid the design of therapeutic agents for the treatment of the complications of diabetes.

Role of Redox Metals, Oxidative Protein Products and Antioxidant Potentials of Thiols in Diabetic Retinopathy

“…A range of low molecular mass thiols have been shown to inhibit dicarbonyl adduction and crosslinking of the thiol-free protein lysozyme, consistent with these thiols being alternative (sacrificial) targets of glycation. Some of these thiols are more efficient modulators of glycation than the established glycation inhibitors such as amino-guanidine (28). A significant decrease in the levels of total proteins can be attri buted to the in cre ased formation of AOPP and decreased levels of GSH and total thiols.…”

Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase

“…Another demerit of these colorimetric assays is that analysis is usually carried out under alkaline condition (pH 9) [20,38], which is far more alkaline than that of human physiological systems. In order to be closer to the physiological condition, the current study mainly employed pH 7.4 reaction medium.…”

“…In clinical examinations, ele-vation of CK levels is an important indicator of damage to muscle and measurement of CK in blood has been used as a diagnostic test for screening suspected muscle diseases [18,19]. Recently, it was reported that the activity of CK was reduced or even lost when incubated with different concentrations of MGO under physiological conditions in vitro [20,21]. Hence, it is reasonable to use aminoguanidine (AG), a typical scavenger for reactive carbonyl compounds [22], to save the active cysteine (Cys) residues in CK by reacting with MGO.…”

“…Conventional methods adopted for measuring CK activity are colorimetric assays and CK activity was mainly determined by monitoring the proton generation during the reaction of ATP and creatine catalyzed by CK with the indicator thymol blue at 597 nm [20,23]. In addition, in clinical laboratories, CK level proportionate to its activity was generally tested based upon enzyme coupled reactions, in which the ATP (or ADP) amount in the system can be reflected by measuring the absorption of produced NADPH (or NADH) at 340 nm [24,25].…”

Oksidatif Stresin Bir Göstergesi Olarak Protein Thioller

“…The finding that thiols as facile targets of glycation and low molecular mass thiols as potent glycation inhibitors, may assist the purpose of therapeutic agents for the treatment of the complications of diabetes 41 . Raised glucose levels can cause oxidative stress in gestational diabetes (GDM) mothers.…”