Identification of Covalent Binding Sites of Phthalic Anhydride in Human Hemoglobin

Abstract

Phthalic anhydride (PA) is a reactive low molecular weight compound used in the chemical industry. The exposure of PA may lead to work-related airway diseases such as rhinitis, chronic bronchitis, and asthma. The exposure gives rise to an increase in hapten-specific IgG antibodies in workers but with a low presence of specific IgE antibodies. In this study, the binding of PA to human hemoglobin (Hb) in vitro was investigated. Trypsin and Pronase E digestion, LC, LC/MS/MS, GC/MS analysis, and nanoelectrospray hybrid quadrupole time-of-flight MS were used to identify the adducted amino acids of the synthesized PA-Hb conjugates. In the conjugate with the molar ratio 1:0.1, a total of six adducted amino acids were identified. N-Terminal valine was found adducted in both the alpha- and the beta-chains as well as a total of four lysines, Val 1, Lys 16, and Lys 61 on the alpha-chain and Val 1, Lys 66, and Lys 144 on the beta-chain. Two types of lysine adducts were found, a phthalamide and a phthalimide. It was also found that PA differs in its binding site as compared to hexahydrophthalic anhydride. The result of this study suggests several interesting applications of biological monitoring.

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“…No N-acetyl-l-lysine adducts were observed at the physiological pH used in our study. In addition, using nanoES-QqTOF we did not find any adducts on the N-terminal valines of Hb, which is known to form adducts with electrophiles [21,23,28]. This is in contrast to Gerberick et al [15] who found adducts between ethyl acrylate or 2-hydroxyethyl acrylate and the amino group of lysine.…”

“…The fractions were evaporated to dryness and dissolved in 20 l of water. An aliquot of 7 l was acidified with 2.5 l of 2.5% TFA, and further purified using C 18 -zip tips [23] prior to analysis using nanoES-QqTOF. The TOF spectra were obtained between m/z 100 and 2000 and product ion scan spectra were obtained between m/z 100 and 1600.…”

“…Visualizing the nanoLC-QqTOF data as 2D images using MSight facilitated the search for the adducted peptides by comparing trypsin digested conjugates and Hb-blank samples. In previous work we have used more time consuming strategies such as acid hydrolysis of the different fractions followed by analysis of the released adducts or manual comparison [21,23,26].…”

“…The b-and/or y -series ions and immonium ions were the most prominent fragment series ions observed, and these pinpointed adducts between the amino acid and acrylates. The determination of the sequence and adducted amino acid was done as described by Jeppsson et al [23]. Several of the ions in the trypsin digested conjugates differing from the Hb-blank samples did not correspond to adducted peptides.…”

“…It has previously been shown that Hb adducts can be used as long-term biomarkers of sensitizing compounds [17,18]. Protein adducts formed by sensitizing chemicals have been characterized in several studies [17,[19][20][21][22][23] using LC/MS/MS or GC/MS. However, the identity of covalent binding sites of acrylates in proteins has not previously been studied.…”