Intra- and intermolecular reactions of 4,4'-diisocyanatodiphenylmethane with human serum albumin.
Diisocyanates are highly reactive industrial chemicals that have been shown to possess toxic activity, including potential for allergic sensitization. To assist in diagnosis of sensitization, immunoassays for diisocyanate-specific antibodies are performed; such assays require preparation of diisocyanate-containing hapten-protein conjugates. Conditions were investigated for formation of conjugates yielding varying degrees of hapten binding. Relative concentrations of haptens and proteins were varied as were pH, temperature, and time of reaction. Quantitation of 4,4'-diisocyanatodiphenylmethane (MDI) binding with human serum albumin (HSA) was assessed by absorbance of the isolated conjugates at 250 nm after determination of the molar extinction coefficient for MDI. At pH 7.4 and 37 degrees C, the binding reaction was found to be biphasic with binding of 5-6 mol of MDI groups/mol of HSA within the first minute, followed by incorporation at a rate of 0.16 mol/min during the next 2 h. Evaluation of reaction products using SDS-PAGE revealed extensive inter- and intramolecular cross-linking of HSA by MDI. Intramolecular cross-linking was accompanied by an increased migration of conjugates from an initial molecular mass of 66 kDa, typical of HSA, to a molecular mass of 44 kDa. The change in migration was also produced by using disuccinimidyl tartarate (DST) as hapten and was eliminated when DST was cleaved with sodium periodate. It was attributed to altered protein shape. Conditions that favored binding of MDI with HSA were a high relative concentration of MDI:HSA, a pH of 9.4, and a temperature of 37 degrees C. Under such conditions it was calculated that 53 mol of MDI were bound per mole of HSA after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Jin
R Z
RZ
Jilin Provincial Institute of Industrial Health and Occupational Disease, Changchun, Jilin Province, People's Republic of China.
Karol
M H
MH
D003485:Cyanates / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D017953:Isocyanates; D007700:Kinetics; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D001559:Benzhydryl Compounds
D003485:Cyanates; D005998:Glycine; D017953:Isocyanates; D012709:Serum Albumin; C005969:4,4'-diphenylmethane diisocyanate; D001559:Benzhydryl Compounds