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J. Biol. Chem., Vol. 279, Issue 8, 6235-6243, February 20, 2004

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Identification of Formaldehyde-induced Modifications in Proteins

REACTIONS WITH MODEL PEPTIDES^*

Bernard Metz, Gideon F. A. Kersten, Peter Hoogerhout, Humphrey F. Brugghe, Hans A. M. Timmermans, Ad de Jong¶, Hugo Meiring¶, Jan ten Hove¶, Wim E. Hennink, Daan J. A. Crommelin, and Wim Jiskoot||

From the Unit Research and Development, The Netherlands Vaccine Institute, 3720 AL Bilthoven, The Netherlands, Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands, and ^¶Laboratory for Analytical Chemistry, National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands

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 chromatography-mass 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.

Received for publication, September 29, 2003 , and in revised form, November 18, 2003.

^* This study was supported, in part, by Grant 9802.086.0/3170.0039 from the "Platform Alternatieven voor Dierproeven" (the Dutch platform on alternatives to animal experiments). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Sorbonnelaan 16, P. O. Box 80082, 3508 TB Utrecht, The Netherlands. Tel.: 31-30-253-6970; Fax: 31-30-251-7839; E-mail: W.Jiskoot{at}pharm.uu.nl.

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