Characterization and comparison of methylene diphenyl diisocyanate haptenated human serum albumin and hemoglobin.
Mhike-M; Hettick-JM; Chipinda-I; Simoyi-RH; Siegel-PD
Toxicologist 2013 Mar; 132(1):307
Methylene diphenyl diisocyanate (MDI) is widely used as a cross-linking agent in the manufacture of polyurethane products. Exposure to diisocyanates (dNCO), such as MDI, is known to cause occupational asthma. MDI haptenation of proteins is central to dNCO immunological sensitization, however, the resultant protein conjugates are complex and difficult to characterize. The objective of the present study was to characterize hemoglobin (Hb) and human serum albumin (HSA) following conjugation to different molar concentrations of MDI. MDI-protein conjugates were acid digested to obtain free methylene dianiline (MDA). MDA was extracted, derivatized with fluorescamine and analyzed by HPLC-fluorescence. MDI-Hb was also digested with trypsin and specific amino acid conjugation sites determined by ultra-performance liquid chromatography-quadruple-tandem time-of-flight mass spectrometry. The trinitrobenzene sulfonic acid assay (TNBS) and denaturing gel electrophoresis were used to determine the extent of cross-linking. MDI conjugation was observed to be dependent on the MDI: protein ratio and the concentration of protein. Greater binding to HSA than Hb was observed and MDI bound to only eight binding sites on Hb compared to twenty for HSA (at 40:1 molar ratio of MDI: protein). Self-polymerization of MDI onto protein was observed on some amino acids at higher MDI concentrations. The TNBS assay was used to confirm cross linking in MDI-HSA with approximately 60% loss of amine reactivity at 40:1 MDI: HSA. More cross-linking was observed at 0.5 mg/ml HSA than at 5mg/ml at 40:1 MDI: HSA It is concluded that MDI has a greater reactivity toward HSA than Hb with respect to the number of residues haptenated and amount of MDI bound per mole of protein.
Toxicology; Methyl-compounds; Methanes; Isocyanates; Polyurethane-foams; Bronchial-asthma; Antibody-response; Proteins; Immune-reaction; Sensitization; Hemoproteins; Serology; Blood-serum; Anilines; Amino-acids; Bioassays; Polymerization; Chemical-binding; Amines
Healthcare and Social Assistance
The Toxicologist. Society of Toxicology 52nd Annual Meeting and ToxExpo, March 10-14, 2013, San Antonio, Texas