Identification and quantification of in vitro adduct formation between protein reactive xenobiotics and a lysine‐containing model peptide

Abstract

Formation of in vitro adducts between different classes of xenobiotics and the lysine-containing peptide Lys-Tyr was monitored by high-performance liquid chromatography and electrospray ionization mass spectrometry. The molecular structures of the main resulting products could be sensitively analyzed by mass spectrometry (flow injection analysis), enabling the detection of characteristic binding formations. Aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde were shown to form stable linkages to lysine amino groups via Schiff bases. Other electrophilic substances (e.g., toluene-2,4-diisocyanate, 2,4-dinitro-1-fluorobenzene, 2,4,6-trinitrobenzene sulfonic acid, dansyl chloride, and phthalic acid anhydride) also formed covalent adducts with lysine residues. The reactivity of the compounds was quantified by measuring the amount of peptide that remained unchanged after incubation for a certain period with the xenobiotic. Although reactivity levels within this group of aldehydes varied only to a small extent, as would be expected, extreme differences were seen among the structurally heterogeneous group of nonaldehyde xenobiotics. These results support the hypothesis that simple chemical reactions may lead to the adduction of nucleophilic macromolecules such as peptides or proteins. Such reactions, in particular, Schiff base formation of aldehydes, have previously been shown to be capable of specifically interfering with costimulatory signaling on T cells. Our results suggest that electrophilic xenobiotics of other classes may also inherit the capacity to exert similar effects. Forming covalent linkage to peptides may represent a possible molecular mechanism of electrophilic xenobiotics in vivo, yielding immunotoxic effects. The model utilized in this study is appropriate for monitoring the adduction of xenobiotics to basic peptides and for analyzing the resulting molecular structures.

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

“…However, in the case of F-ALDH, the substrate specificity suggested an independent response on the type of aldehyde (aliphatic or aromatic) used in the reaction (Figure 4). Also, the major difference observed for the catalysis of benzaldehyde and the 2-isopropyl and 4-isopropil derivates of this aromatic substrate (Figure 4) is in accordance with the specificity variation within the structurally heterogeneous group of benzaldehyde compounds [67]. In this sense, further 3D modeling and molecular docking analyses could highlight subtle structural features responsible for the substrate specificity of both substrates and cofactors used by this cold-active ALDH.…”

Chemical Reactivity and Skin Sensitization Potential for Benzaldehydes: Can Schiff Base Formation Explain Everything?

“…The other one is a triplet (J = 6.8 Hz) at 3.45 ppm. Their respective 13 C NMR shifts extracted from a HSQC experiment 20 are 127.2 ppm for the aromatic proton pair and 39.9 ppm for an aliphatic methylenic group. In the HMBC experiment 21 depicted in Figure 5, these two proton groups share 3 J C,H couplings to a common carbon atom resonating at 171.1 ppm.…”

“…In parentheses is shown the sensitization potential as an EC3 value in the LLNA or, if available, other information regarding the skin sensitization potential. For 13,18,and 19, no data regarding the skin sensitization potential are available, but these chemicals were included to complete the SAR study for reactivity. reported as a classical nonsensitizer based on animal tests but was recently discovered to be a sensitizer in a human repeat insult patch test (HRIPT; RIFM, personal communication).…”

“…12 These chemicals only promoted the formation of disulfide bridges at the Cys residue. However, under much more concentrated reaction conditions (80 mM peptide and 200 mM test chemical), Reichhardt et al 13 reported Schiff base formation with the ε-NH 2 -group of Lys-Tyr for the two aldehydes formaldehyde and benzaldehyde. Furthermore, supplementary information from Aleksic et al 14 reports Schiff base formation in a peptide reactivity assay for cyclamen aldehyde, hydroxycitronellal, benzaldehyde, and αhexyl cinnamic aldehyde.…”

New analytical strategies in studying drug metabolism

“…Reichardt et al proposed a model using a dipeptide for monitoring adduct formation of xenobiotics and for characterization of molecular structures [74]. Lysine-tyrosine (Lys-Tyr) was chosen as model peptide.…”