The Reaction of Formaldehyde with Proteins. V. Cross-linking between Amino and Primary Amide or Guanidyl Groups

Fraenkel‐Conrat, H.; Olcott, Henry Steel

doi:10.1021/ja01188a018

Cited by 364 publications

(159 citation statements)

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“…In contrast to previous studies with amino acids (18,19), intermolecularly cross-linked peptides were not detected. This may be because of differences in reaction conditions and possible lower reactivity of peptides as compared with free amino acids.…”

Section: Scheme 2 Reduction Of Primary Amino Groups By Adding Formalcontrasting

confidence: 99%

Identification of Formaldehyde-induced Modifications in Proteins

Metz

Kersten²,

Hoogerhout³

et al. 2004

Journal of Biological Chemistry

513

307

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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...

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Section: Scheme 2 Reduction Of Primary Amino Groups By Adding Formalcontrasting

confidence: 99%

Identification of Formaldehyde-induced Modifications in Proteins

Metz

Kersten²,

Hoogerhout³

et al. 2004

Journal of Biological Chemistry

513

307

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“…The chemical complexity of formaldehyde-mediated reaction products was appreciated 70 years ago (5). Initially using amino acids, and subsequently proteins and other substrates, it was shown that formaldehyde reacts in vitro with a wide range of functional groups, forming a complex array of products (6,7). It has been known since the 1940s that such products can include intramolecular and intermolecular crosslinked species and that the reaction conditions (e.g.…”

Section: Basic Chemistrymentioning

confidence: 99%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

311

261

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Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.Prior to its use in the chromatin field, formaldehyde use had a long history in a number of fields, including vaccine production (1, 2) and histology (3). In this review, we focus on its use in chromatin immunoprecipitation approaches and protein-protein interaction studies applied to understand the location and abundance of transcription factor binding along DNA. A recent complementary perspective highlights gaps in knowledge with a particular focus on how formaldehyde crosslinking data have been used to interpret aspects of chromatin three-dimensional organization (4). Here, we briefly review prior work describing formaldehyde reactivity toward proteins, DNA, and their constituent monomers. This information provides a basis for understanding how formaldehyde functions in widely used assays in the chromatin field, and conversely, highlights less well understood aspects of formaldehyde behavior in cells. These issues are of significance for designing crosslinkingbased studies as well as for properly interpreting the resulting data. The analysis of formaldehyde-fixed chromatin has provided fundamental insights into where and when regulatory factors associate with the DNA template in vivo, but it in general does not provide unambiguous information about chromatin binding kinetics. A major goal of ongoing work is to understand kinetic and thermodynamic aspects of chromatin complex assembly at single copy loci in vivo. Development of experimental strategies to achieve these goals will require a deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells.The following discussion provides a framework for understanding aspects of formaldehyde function when used to trap macromolecular complexes in cells, with the main features shown in Fig. 1. Beginning with basic chemical reactivity, this review will explore the ability of formaldehyde to crosslink with proteins and DNA to form protein-protein or protein-DNA complexes, common molecular quenchers, and the potential for crosslink reversal. Progress in capturing crosslinked complexes will also be discussed with an emphasis on the impact of a better understanding of formaldehyde chemistry in vivo. Basic ChemistryFormaldehyde is the smallest aldehyde, an electrophilic molecule susceptible to chemical attack by a wide range of nucleophilic species of ...

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“…As the VP 28 gene sequence inserted in pET28a vector (Novagen) contained only the hydrophilic part, the protein obtained was in the soluble form. In addition, the protein might have undergone cross-linking in the presence of formalin during tissue fixation [9], necessitating antigen retrieval [20,21]. Preparation of IVP that involved addition of 0.2% formalin (v/v) might have resulted in cross-linking of the VP28 epitopes on the virus.…”

Section: Wssv-vaccines and Immunohistochemistrymentioning

confidence: 99%

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon

Kulkarni

Rombout

Singh

et al. 2013

Fish & Shellfish Immunology

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a b s t r a c tSeveral oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

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The Reaction of Formaldehyde with Proteins. V. Cross-linking between Amino and Primary Amide or Guanidyl Groups

Cited by 364 publications

References 0 publications

Identification of Formaldehyde-induced Modifications in Proteins

Identification of Formaldehyde-induced Modifications in Proteins

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: An uptake and processing study in the midgut of Penaeus monodon

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