Identification of Formaldehyde-induced Modifications in Proteins

Abstract

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatographymass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.Aldehydes, such as formaldehyde and glutaraldehyde are widely employed reagents in the biochemical, biomedical, and pharmaceutical fields. Formaldehyde, for example, is applied to inactivate toxins and viruses for the production of vaccines, such as diphtheria, tetanus toxoid, hepatitis A, anthrax, and inactivated polio vaccine, and to stabilize recombinant pertussis toxin (1-4). The vaccine quality depends to a considerable extent upon the chemical modifications caused by the formaldehyde treatment (1, 5, 6). Formaldehyde is also used for isotope-labeling of proteins (7-9), for studying protein-protein interactions, e.g. histone organization in nucleosomes (10 -12), and for fixation of cells and tissues (13). Glutaraldehyde is utilized for the preparation of bioprostheses such as heart valves and vascular grafts (14 -16) and for conjugation of enzymes to carrier systems (17). These examples demonstrate the wide range of roles of aldehydes in the biomedical field. Besides the use of aldehydes in diverse applications, they can also destroy important sites of pro...

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

“…2). In this case the apparent loss of four hydrogens can be ascribed to the formation of two methylene bridges in an intramolecular cross-link that probably involves the Lys 4 and the N-terminal arginine [16] (Fig. 2).…”

“…The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically to fix tissues and cells and it is known as a cross-linking agent involving a wide set of amino acid residues [14,15]. Metz et al reported a systematic investigation on a series of synthetic peptides in the presence of glycine or NaCNBH 3 [16,17]. When the reducing agent is added to the mixture after 24 h incubation with formaldehyde, besides the expected dimethylated peptide a secondary product featuring 2 mass units less can be observed whereas a Schiff-base is generated at the N-termini.…”

Influência da adição de plastificante do processo de reticulação na morfologia, absorção de aguá e propriedades mecânicas de filmes de alginato de sódio

“…Reticulantes, como o formaldeído, têm sido largamente utilizados para induzir modificações em proteínas e polissacarídeos. Exemplos incluem a reticulação da quitosana via formação de N-metileno-quitosana 17 ; reticulação da blenda alginato de sódio e hidroxietilcelulose com glutaraldeído em meio ácido seguido de um segundo processo de reticulação com uréia-formaldeído e ácido sulfúrico 18,19 ; reticulação por formaldeído e epicloridrina de açúcares obtidos da polpa de beterraba 20 ; formação de pontes estáveis de metileno em modelos de peptídeos 21 ; reticulação de colágeno 22 e proteína de semente de algodão 23,24 ; efeito sobre as propriedades mecânicas, permeabilidade ao vapor de água e solubilidade de glúten 25 , gliadina 26,27 , proteína de amendoim 28 , filmes de proteína isolada de soja 29,30 e, ainda, modificações em filmes de gelatina 31 . Neste estudo, os efeitos da adição de sorbitol como plastificante e do processo de reticulação com formaldeído, nas propriedades de filmes de alginato de sódio e alginato de sódio/sorbitol, foram investigados.…”

Formaldehyde degradation by a newly isolated fungus Aspergillus sp. HUA

“…It is often released from wastewater of construction, textile, wood processing, furniture, and pharmacy industry (Tang et al 2009). Formaldehyde is a water-soluble compound, which can diffuse into many tissues rapidly, react with different macromolecules such as proteins and nucleic acids, cause DNA-DNA, protein-DNA, and protein-protein cross-links (Metz et al 2004;Merk and Speit 1998;Solomon and Varshavsky 1985). Therefore, it has a toxic effect on all organisms, the IARC (2006) (International Agency for Research on Cancer) has classified formaldehyde as a human carcinogen that causes nasopharyngeal cancer and probably leukemia.…”