muramidase and 2-4-dinitrophenylhydrazine

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

Incubation of model proteins with the toxic lipid peroxidation product malondialdehyde resulted in a time- and concentration-dependent increase in carbonyl contents. Carbonyl groups were detected either spectrophotometrically or immunochemically after derivatization with 2,4-dinitrophenylhydrazine. Although significant adduction occurred when modifications were performed at pH 7.0, carbonyl formation was most extensive when modifications were carried out at pH 4.0 or pH 5.0. Similarly, formation of intermolecular crosslinks was most extensive when reactions were carried out under mildly acidic conditions. Our results raise the possibility that malondialdehyde adducts contribute to the carbonyl content of proteins recovered from mammalian tissues.

Topics: Animals; Catalase; Endopeptidase K; Humans; Immunohistochemistry; Kinetics; Lipid Peroxidation; Malondialdehyde; Mammals; Models, Biological; Muramidase; Phenylhydrazines; Proteins; Serine Endopeptidases; Serum Albumin; Serum Albumin, Bovine; Spectrophotometry

Protein carbonyls are reported to increase in aging and in pathologies such as Alzheimer's disease and ischemic injury. Detailed study of this important issue has, however, been hampered by lack of an appropriate method to identify individual carbonylated proteins. We describe here an immunoblot method to identify individual carbonylated proteins. We describe here an immunoblot method to investigate protein carbonyls reactive to 2,4-dinitrophenyl hydrazine. Rabbit polyclonal antibodies against 2,4-dinitrophenyl hydrazine were used to study the proteins derivatized by the reagent in one- or two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting. More than 25 proteins with high carbonyl contents were clearly demonstrated in two-dimensional immunoblot of rat tissue soluble proteins. The method could detect concentrations as low as 1 pmol of carbonyls. The signals were mostly abolished by prior treatment of tissue proteins with sodium borohydride to reduce carbonyls. Fragments generated by V8 protease digestion of a single protein exhibited signal intensities of varying degrees, indicating that carbonylation is not uniform in different amino acid sequences. Proteins treated with glucose or aldehydes gave rise to positive signals, suggesting that the finding of carbonyls in tissue proteins is not necessarily an indication of direct oxidation of side chains of amino acid residues.

Topics: Acetaldehyde; Animals; Antibodies; Antibody Specificity; Brain Chemistry; Catalase; Chickens; Electrophoresis, Gel, Two-Dimensional; Glucose; Immunoblotting; Indicators and Reagents; Male; Muramidase; Oxidation-Reduction; Peptide Fragments; Phenylhydrazines; Proteins; Rabbits; Rats; Rats, Inbred F344; Serum Albumin, Bovine