| Popis: |
The molecular masses of the bacterially expressed recombinant HuAChE and its conjugates with series of alkyl methylphosphonofluoridates and with diisopropyl fluorophosphate (DFP), were measured by electrospray - ionization mass spectrometry (ESMS). The mass of HuAChE (measured as 64700 Da, calc. 64695 Da) increased, following reactions with sarin, isobutyl methyl phosphorofluoridate (IBMPF), 1, 2-dimethylpropyl methyl phosphorofluoridate (DMPF), soman and DFP, by 120, 140, 150, 160 and 160 Da respectively. These values were in excellent agreement with the calculated masses of the adducts and reflected both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of the phosphyl adducts change with time to yield a common product with molecular mass of 64780 Da, which is consistent with dealkylation of the phosphonyl moieties. By sequential ESMS measurements we were able to estimate the kinetics of evolution of the aged product of the HuAChE-soman adduct (t1/2 ≈ 50 sec, at pH 6.0). This rate is in good agreement with that determined by kinetic measurement of the development of non-reactivability under similar conditions. It is important to note that in agreement with the accepted mechanism of dealkylation, only two molecular species are evident in the sequential ESMS measurements during the aging process. |
