Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

doi:10.1016/j.ab.2003.10.041

Analytical Biochemistry

Volume 325, Issue 2, 15 February 2004, Pages 326-336

https://doi.org/10.1016/j.ab.2003.10.041 Get rights and content

Abstract

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio ∼50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20–30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20–30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S₂O₃)₂]³⁻ resulted in formation of the adducts HSA + Au(S₂O₃) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S₂O₃)₂]³⁻ blocked the formation of gold adducts.

Section snippets

Materials

MilliQ water (Millipore) was used in all experiments. Organic solvents, acetic acid, and ammonium acetate were from Asia Pacific Specialty Chemicals. d,l-Dithiothreitol (DTT) and iodoacetamide were purchased from ICN Biochemicals (OH). Cysteine, glutathione, sodium bis(thiosulfato)gold(I), and fatty acid-free human serum albumin were purchased from Sigma–Aldrich (MO). Sephadex G-25 (medium) was from Amrad-Pharmacia.

Plasma collection

Blood (∼5 ml) was collected by venepuncture from nine healthy volunteers into

Optimal conditions for acquisition of ESI mass spectra of human serum albumin

In preliminary experiments, attempts were made to acquire high-quality (high signal-to-noise; high-resolution) positive-ion ESI mass spectra of commercial human serum albumin using a Micromass Q-TOF2 mass spectrometer. Under many experimental conditions where instrument parameters and solution conditions were varied, it was not possible to obtain ESI mass spectra of commercial HSA of sufficient quality to enable resolution between ions from unmodified HSA and ions from modified HSA (e.g.,

Conclusions

The abundance of HSA in plasma enabled its analysis using ESI–MS by direct injection into the mass spectrometer of fresh plasma diluted 600-fold. These experiments strongly support that ∼25% of circulating HSA molecules contain bound cysteine. The adducts HSA + Au(S₂O₃) and HSA + Au could be readily detected after in vitro reaction of HSA with [Au(S₂O₃)₂]³⁻. Furthermore, blocking experiments using iodoacetamide strongly support the binding of gold to Cys34.

The ability to obtain highly resolved ESI

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Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

Abstract

Section snippets

Materials

Plasma collection

Optimal conditions for acquisition of ESI mass spectra of human serum albumin

Conclusions

J. Biol. Chem.

Adv. Prot. Chem.

J. Mol. Biol.

Pathophysiology

J. Chromatogr. B

Toxicol. Appl. Pharmacol.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Anal. Biochem.

Biochim. Biophys. Acta

Diabetes Res. Clin. Pract.

All About Albumin: Biochemistry, Genetics and Medical Applications

Involvement of a lysine residue in the N-terminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+

Eur. J. Biochem.

Practical aspects of the ligand-binding and enzymatic properties of human serum albumin

Biol. Pharmaceut. Bull.

Crystal structure of human serum albumin at 2.5 Å resolution

Protein Eng.

Involvement of a lysine residue in the N-terminal Ni²⁺ and Cu²⁺ binding site of serum albumins. Comparison with Co²⁺, Cd²⁺ and Al³⁺