Elsevier

Biochemical Pharmacology

Volume 79, Issue 5, 1 March 2010, Pages 784-791
Biochemical Pharmacology

Reaction of human albumin with aspirin in vitro: Mass spectrometric identification of acetylated lysines 199, 402, 519, and 545

https://doi.org/10.1016/j.bcp.2009.10.007Get rights and content

Abstract

The aspirin esterase activity of human plasma is due to butyrylcholinesterase and albumin. Our goal was to identify the amino acid residues involved in the aspirin esterase activity of albumin. Fatty acid-free human albumin and human plasma were treated with aspirin for 5 min–24 h. Acetylated residues were identified by LC/MS/MS and MALDI-TOF/TOF mass spectrometry of tryptic peptides. Treatment with 0.3 mM aspirin resulted in acetylation of Lys-199, Lys-402, Lys-519, and Lys-545. Treatment with 20 mM aspirin resulted in acetylation of 26 lysines. There was no acetylation of Tyr-411, under any conditions. Acetylated lysine was stable for at least 21 days at pH 7.4, 37 °C. Albumin acetylated by aspirin had reduced esterase activity with β-naphthyl acetate as shown on gels stained for esterase activity. It was concluded that the aspirin esterase activity of albumin is a pseudo-esterase activity in which aspirin stably acetylates lysines and releases salicylate.

Introduction

Hawkins et al. reported that aspirin transfers its acetyl to the ɛ-amino group of lysine residues of albumin both in vitro[1] and in vivo[2]. Later the labeled residue was identified as Lys-199 [3]. The crystal structure of human albumin confirmed that aspirin acetylates Lys-199 [4]. To date no other acetylated residues have been identified, though up to 6 additional radiolabeled peptides were found on the radioautograph of the peptide map of aspirin-treated albumin [2].

Albumin is regarded as an aspirin esterase because salicylic acid is produced by incubation of aspirin with albumin [5]. The esterase activity of albumin with p-nitrophenyl acetate is predominantly a pseudo-esterase activity in which up to 59 lysines are stably acetylated. In addition, Tyr-411 of human albumin is rapidly acetylated by p-nitrophenyl acetate (t1/2 = 0.56 min) and slowly deacetylated (t1/2 = 61 h) [6], [7]. The three goals of our study were to determine whether the aspirin esterase activity of albumin is also a pseudo-esterase activity resulting in stable acetylation of many residues; if so, to identify the acetylated residues by mass spectrometry; and to determine whether Tyr-411 is involved in the reaction of albumin with aspirin.

Section snippets

Materials

A 1 mg/ml solution of fatty acid-free human albumin (Fluka 05418, via Sigma–Aldrich, St. Louis, MO) was prepared in 100 mM potassium phosphate buffer, pH 7.4. The amino acid sequence for the albumin from this source is given in accession number gi:122920512. This albumin has Glu-396 in peptide QNCELFE*QLGEYK where other albumin sequences have Lys-396. A 1 mg/ml solution of porcine pepsin (Sigma–Aldrich P6887) in 10 mM HCl, as well as 20 μg of sequencing grade modified trypsin (Promega V5113,

Residues acetylated by 0.3 mM aspirin

Two different mass spectrometry methods (MALDI-TOF/TOF and Q-TRAP MS/MS) were applied to identify tryptic peptides of albumin acetylated by aspirin. A 24-h reaction of 15 μM human albumin with 0.3 mM aspirin at 37 °C resulted in the acetylation of four albumin residues, namely Lys-199, Lys-402, Lys-519, and Lys-545. No additional acetylation sites were found when the physiological concentration of albumin (600 μM) was used. The masses of labeled tryptic peptides were increased by +42 m/z due to

Covalent modification of lysines by aspirin

Aspirin transfers its acetyl group to human albumin in vitro. This interaction involves multiple acceptor sites, identified as lysine residues by two different mass spectrometry methods (MALDI TOF/TOF and Q-TRAP). We conclude that the observed adducts are due to covalent binding of the acetyl group to ɛ-N-amino groups of lysines. Non-covalent adducts do not survive the MALDI process due to the low pH of the matrix as well as the heat generated by high laser energy [14]. Furthermore,

Acknowledgements

Mass spectra were obtained with the support of the Mass Spectrometry and Proteomics core facility at the University of Nebraska Medical Center. This work was supported by a grant from the US Army Medical Research and Materiel CommandW81XWH-07-2-0034 and an NCI Cancer Center Support GrantCA36727. M.L. was awarded a Fulbright Russia student grant.

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