Determination of hexahydrophthalic anhydride adducts to human serum albumin.
Hexahydrophthalic anhydride (HHPA) is a highly sensitizing industrial chemical that is known to covalently bind to endogenous proteins. The aim of this study was to determine the binding sites of HHPA to human serum albumin (HSA). Conjugates between HSA and HHPA, at two different molar ratios, were synthesized under physiological conditions. The conjugates were digested with trypsin and Pronase E to obtain specific peptides and amino acids, which were separated by liquid chromatography (LC). Fractions containing modified peptides were detected through quantification of hydrolysable HHPA using LC coupled to a triple quadrupole mass spectrometer with electrospray ionization. Modified residues in albumin were identified by sequence analyses using nanoelectrospray quadrupole time-of-flight mass spectrometry. A total of 36 HHPA adducts were found in the HSA-HHPA conjugate with 10 times molar excess of added HHPA. In the conjugate with a molar ratio of 1:0.1 of added HHPA, seven HHPA adducts were found bound to Lys(137) (domain IB), Lys(190), Lys(199) and Lys(212) (domain IIA), Lys(351) (domain IIB), and Lys(432) and Lys(436) (domain IIIA). Moreover, several of these adducted albumin peptides were detected in nasal lavage fluid from one volunteer exposed to HHPA. The binding sites of HHPA to HSA have been determined, thus identifying potential allergenic chemical structures. This knowledge generates the possibility of developing methods for the biological monitoring of HHPA exposure by analysing tryptic peptides including these binding sites.
Kristiansson
Monica H
MH
Department of Occupational and Environmental Medicine, Institute of Laboratory Medicine, University Hospital, SE-221 85 Lund, Sweden. monica.kristiansson@ymed.lu.se
Lindh
Christian H
CH
Jönsson
Bo A G
BA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D004781:Environmental Exposure; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010796:Phthalic Anhydrides / Q000737:chemistry* / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010796:Phthalic Anhydrides; D012709:Serum Albumin; D014357:Trypsin; D011402:Pronase; D008239:Lysine