Cyanide Adducts with Human Plasma Proteins:  Albumin as a Potential Exposure Surrogate

Abstract

Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

“…In addition, we conducted an experiment to assess the involvement of Cys34 in covalent binding, in which HKI-272 was incubated in human plasma with/without coexistence of iodoacetamide (5 mM), and the recovery was similar in both conditions. Fasco et al (2007) observed the formation of CN-Cys34 adduct on treatment of HSA with CN. They proposed that Cys34 sulfhydryl moiety should exist as a disulfide bond before reacting with CN because CN could not react with a free sulfhydryl group (Fasco et al, 2007).…”

“…Fasco et al (2007) observed the formation of CN-Cys34 adduct on treatment of HSA with CN. They proposed that Cys34 sulfhydryl moiety should exist as a disulfide bond before reacting with CN because CN could not react with a free sulfhydryl group (Fasco et al, 2007). However, Bertucci et al (1998) reported the derivatization of the Cys34 in human albumin by ethacrynic acid using mass spectrometry and circular dichroism, which suggests that Cys34 is a free sulfhydryl.…”

Cyanides in the environment—analysis—problems and challenges

“…Beside the spectrophotometric and chromatographic techniques, chemiluminescence (Goi et al 2007) or capillary electrophoresis is used, however not so often, in the analysis of environmental samples (Fasco et al 2007; Sadeg and Belhadj-Tahar 2009). Mass spectrometry with ionization of selected ions in stream (SIFT-MS) is used particularly in the determination of HCN in the exhaled air.…”

Development of a Fluorescence-Based Sensor for Rapid Diagnosis of Cyanide Exposure

“…For HSA, the elevated fluorescence may have been due to the release of cyanide from cyanide–HSA adducts under acidic conditions. 37 …”