title: Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry.
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 approximately 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(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.
authors.affiliation: Department of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia. jennifer_beck@uow.edu.au
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Beck
authors.forename: Shanika
authors.initials: S
authors.lastname: Ambahera
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Yong
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Sheil
authors.forename: John
authors.initials: J
authors.lastname: de Jersey
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Ralph
mesh_terms: D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol; D005978:Glutathione / Q000378:metabolism; D017612:Gold Compounds; D006801:Humans; D007460:Iodoacetamide; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D017612:Gold Compounds; D012709:Serum Albumin; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D007460:Iodoacetamide