(294c) 2,4-Toluene Diisocyanate Adducted Peptides in Conjugates Formed with Human Serum Albumins in Vitro and in Vivo

Among workers of toluene diisocyanates (TDI) exposure, specific immune responses were reported to be related to the hypersensitive syndromes. The conjugates of human serum albumin (HSA) in vitro and protein adducts in plasma were reported also. In this study, the adducted albumin of workers in long time exposure were isolated with specific antiserum by affinity chromatograph, and the adducted peptides obtained from trypsin digestion were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis, and tandem mass analysis by MALDI-TOF/TOF. The results were compared with those spectra about tryptic peptides obtained from conjugates synthesized in vitro.

Those plasma collected from forty TDI exposed workers with occupational allergic symptoms, for whom the specific IgG4 and IgE antibodies against 2,4-TDI and TDI-HSA were checked, and those antibody binding abilities of its peptides digested by protease K were examined also. About ten major bands from TDI-HSA lysates were estimated to have molecular mass about 4.9Kda, 3.7kDa and 2.7kDa, within the range from 27.4~2.1kDa. At least six fragments of TDI-HSA and four fragments of HSA were recognized by Ig G4 antibodies on IEF-PAGE, respectively. Peptides below 20 kD were recognized by Ig G4 antibodies also. In TDI-HSA lysates within the range of pI value 6.0 to 6.9, different patterns at more basidic area and serological heterogeneity was observed. From the spectra of synthetic conjugates, fragments of TDI-HSA tryptic digests distinct different from HSA tryptic digests, were identified on Maldi-TOF/TOF experiments, by the appearance of an ions of m/z175 or m/z190 in the MS/MS spectra. The results support that the short time conjugation reaction of isocyanate with albumin is selective for several defined sites of lysine residues. From the results of electrospray mass spectrometry (ESI-MS), about ten peptides ion peaks equivalent to different TDI moiety were observed and possible sequences were elucidated. Two tryptic peptides contain phenylalanine, the reaction sites are Lys 249, 581; one contains tyrosine adducted on Lys375; two contains tyrosine and phenylalanine on Lys 183, 413; peptide adducted on Lys 236 contain phenylalanine and tryptophan.

By comparison of spectra with in vitro experiments, results analyzed from digested conjugates formed in vivo, common sites peptides were found, Lys 249 were verified by MS/MS experiment. All the fragments are primarily b- and y- series, from which the peptide sequence can be easily deduced. The adducted TDI moieties at the ?'-NH2 groups of lysine is noted for the whole molecule ( 174.9 Da) or its saturated isocyanate side chain fragments.

Because of the highly reactive properties, the reaction time is the most important factor for isocyanate compounds. Our results showed the most possible ten binding sites in albumin obtained under physiological condition, and several common sites were found in the plasma of exposurers. The similar adducted locations in the albumin molecule in vitro and in vivo, support the application value of the adductive peptides synthesized.