| Abstract
| - In the present study, we applied for the first time 31P diffusion NMR to resolve different speciesobtained by the addition of organophosphorus compounds (OP) such as diisopropyl phosphorofluoridate (DFP) or 1-pyrenebutyl phosphorodichloridate (PBPDC) to α-chymotrypsin (Cht). 31P diffusionNMR was used since the products of these reactions constitute a mixture of OP-covalent conjugatesof the enzyme and OP-containing hydrolysis products that have noninformative 1H NMR spectra.It was shown that the peak, attributed to the covalent native diisopropylphosphoryl-Cht (DIP-Cht) conjugate by chemical shift considerations, has a greater diffusion coefficient (D = (0.65 ±0.01) × 10-5 cm2 s-1) than expected from its molecular mass (approximately 25 kDa). This peakwas therefore suggested to consist of at least two superimposed signals of diisopropyl phosphoryl(DIP) pools of high and low molecular weights that happen to have the same chemical shift. Thisconclusion was substantiated by the use of DMSO-d6 that separated the overlapping signals.Diffusion measurements performed on the extensively dialyzed and unfolded DIP-Cht conjugatestill resulted in a high diffusion coefficient ((0.30 ± 0.05) × 10-5 cm2 s-1) relative to the assumedmolecular mass. This observation was attributed to a dynamic dealkylation at the OP moiety (i.e.,aging) that occurred during the relatively long diffusion measurements, where DIP-Cht wasconverted to the corresponding monoisopropyl phosphoryl Cht (MIP-Cht) conjugate. Homogeneousaged forms of OP-Cht were obtained by use of DFP and heat-induced dealkylation of DIP-Cht, andby PBPDC that provided the aged form via the hydrolysis of a P-Cl bond (PBP-Cht). The thermallystable aged conjugates enabled a reliable determination of the diffusion coefficients over severaldays of data acquisition, and the values found were (0.052 ± 0.002) × 10-5 cm2 s-1 and (0.054 ±0.004) × 10-5 cm2 s-1 for the MIP-Cht and the PBP-Cht adducts, respectively, values in the rangeexpected for a species with a molecular weight of 25 kDa. The advantages and limitations of 31Pdiffusion NMR in corroborating the type of species that prevail in such systems are briefly discussed.
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