muramidase and pimagedine

Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

Topics: Amino Acid Sequence; Animals; Biological Assay; Catalysis; Cattle; Electrochemistry; Electrodes; Enzyme Inhibitors; Glycated Serum Albumin; Glycation End Products, Advanced; Glycosylation; Guanidines; Humans; Models, Molecular; Molecular Sequence Data; Muramidase; Protein Conformation; Pyruvaldehyde; Serum Albumin; Serum Albumin, Bovine

Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

Topics: Fluorescence; Fructose; Glucose; Glycation End Products, Advanced; Guanidines; Lactoglobulins; Muramidase; Pentetic Acid; Phenylhydrazines; Serum Albumin, Bovine

1. The effects of AL0671, a novel potassium channel opener, on protein glycation and low-density lipoprotein (LDL) oxidation were tested. 2. AL0671 dose-dependently inhibited both fluorescence development of bovine serum albumin and cross-linking of lysozyme. These inhibitory effects for glycation were no less potent than aminoguanidine. 3. AL0671 dose-dependently inhibited both increase in negative charge and apo B-100 fragmentation during incubation of LDL with Cu2+. In addition, AL0671 significantly decreased the LDL degradation in rat peritoneal macrophages. 4. Neither pinacidil nor levcromakalim inhibited protein glycation and LDL oxidation. 5. Antioxidant properties of AL0671 might be due to its potent electron-donating ability, and this agent is expected to be useful for hypertensive diabetes.

Topics: Animals; Aspirin; Bridged Bicyclo Compounds; Electrophoresis; Glycosylation; Guanidines; Humans; Lipoproteins, LDL; Macrophages, Peritoneal; Male; Muramidase; Oxidation-Reduction; Potassium Channels; Rats; Rats, Wistar; Serum Albumin, Bovine

We investigated the effect of exogenously applied 3-deoxyglucosone, a major carbonyl intermediate, on the Maillard reaction. The fluorescence intensity of the product of the reaction of bovine serum albumin with 3-deoxyglucosone was higher than that with an equivalent amount of glucose. Similarly the rate of polymerization of lysozyme in the presence of 3-deoxyglucosone was also greater than with glucose, and collagen incubated with 3-deoxyglucosone was less digestible than collagen incubated with glucose. By contrast, aminoguanidine inhibited an increase in fluorescence of the Maillard compounds and the polymerization of protein, both of which were stimulated by 3-deoxyglucosone. These results suggest that 3-deoxyglucosone accelerates the advanced stage of the Maillard reaction and that aminoguanidine acts on 3-deoxyglucosone to inhibit its action in the advanced stage of the Maillard reaction.

Topics: Collagen; Deoxy Sugars; Deoxyglucose; Glucose; Glycosylation; Guanidines; Maillard Reaction; Microbial Collagenase; Muramidase; Polymers; Serum Albumin, Bovine; Spectrometry, Fluorescence