Observation of Triplet Traces Obtained with Inversion Recovery Method in Both Residual Water- and H2O/D2O-Albumin Mixture by Using 400 MHz Proton NMR

Abstract

H 2 O/D 2 O mixtures are being used as solvent for proteins and some other chemical compounds 1-7 . In the mixture of H 2 O and D 2 O, there are three molecular species H 2 O, D 2 O and HDO in a dynamic equilibrium 8,9 . However, NMR spectrum of the mixture shows a single line at all static magnetic fields. Accordingly, the studies involving such solvents are mainly based on this single spectrum of water. The interpretation of the spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation data in H 2 O/ D 2 O mixture is known to be difficult 8,9 . The analysis of the equilibrium data among H 2 O, D 2 O and HDO was also reported to be difficult 10 . On the other hand, the small spin-spin splittings of H 2 O signal by deuterium (D) in H 2 O/D 2 O were studied by proton NMR for deuteriated water 10 . In addition, a triplet of 1 H NMR signals, which is the splitting of H 2 O signal by deuterium, was recently presented through a special free induction decay (FID) processing programme 11 . The splittings may be demonstrated further by the relaxation effects of the triplet components. The demonstration may be achieved by acquiring inversion recovery (IR) data of the triplet. It should contribute to the interpretation of NMR relaxation mechanisms NMR studies involving H2O/D2O mixtures are mainly based on a single spectrum of water. However, some recent papers have revealed the presence of an HDO triplet in H2O/D2O, which is caused by the splittings of H2O signal by deuterium (D). Observation of inversion recovery (IR) traces of this triplet should be very useful for analyzing relaxation data in such mixtures. In this work, in order to obtain the traces, inversion recovery signal intensities of residual water and a mixture of 0.10 H2O and 0.90 D2O were acquired versus a set of short delay times in the presence or absense of 0.2 g albumin. T1 and T2 curves of the same samples were also obtained by using different sets of much longer times for checking the effect of radiation damping on the traces. Experiments were carried out with a 400 MHz proton NMR spectrometer equipped with a topspin programme for relaxation measurements. Inversion recovery data of the traces and T1 curves were obtained with the inversion recovery method, while T2 curves were obtained with the Carr-Purcell-Meiboom-Gill method. Data was processed through an analysis programme of the topspin. Data showed that radiation damping is effectively reduced upon addition of albumin. Furthermore, the data of the residual water-and the mixture with albumin demonstrated the traces of the triplet at the initial part of a single inversion recovery line. Our results suggest that the splitting of water proton signal by deuterium can be detectable by the inversion recovery method for D2O-and the mixture containing albumin.