NMR resonance splitting of urea in stretched hydrogels: Proton exchange and 1 H/ 2 H isotopologues Philip W. Kuchel a,⇑ , Christoph Naumann a , Bogdan E. Chapman a , Dmitry Shishmarev a , Pär Håkansson c , George Bacskay b , Noel S. Hush a,b a School of Molecular Bioscience, University of Sydney, NSW 2006, Australia b School of Chemistry, University of Sydney, NSW 2006, Australia c School of Chemistry, University of Southampton, SO17 1BJ, United Kingdom article info Article history: Received 3 June 2014 Revised 29 July 2014 Available online 27 August 2014 Keywords: 1 H NMR spectroscopy 2 H NMR spectroscopy [1,3- 15 N]urea Chirality Gelatin gel Isotopomers Residual dipolar coupling Residual quadrupolar coupling Stretched gel abstract Urea at 12 M in concentrated gelatin gel, that was stretched, gave 1 H and 2 H NMR spectral splitting pat- terns that varied in a predictable way with changes in the relative proportions of 1 H 2 O and 2 H 2 O in the medium. This required consideration of the combinatorics of the two amide groups in urea that have a total of four protonation/deuteration sites giving rise to 16 different isotopologues, if all the atoms were separately identifiable. The rate constant that characterized the exchange of the protons with water was estimated by back-transformation analysis of 2D-EXSY spectra. There was no 1 H NMR spectral evidence that the chiral gelatin medium had caused in-equivalence in the protons bonded to each amide nitrogen atom. The spectral splitting patterns in 1 H and 2 H NMR spectra were accounted for by intra-molecular scalar and dipolar interactions, and quadrupolar interactions with the electric field gradients of the gelatin matrix, respectively. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction 1.1. Aims A very high concentration of urea [(NH 2 ) 2 CO; 12 M] in gelatin (1:1 w/w) in water does not prevent the formation of a gel when the temperature is below 25 °C. Thus, the concentration of the amide protons and those of water (plus the exchangeable protons of the carboxyl, amine and amide groups of the protein, gelatin) are of the same order of magnitude. The 1 H NMR spectrum displays a single resonance for urea [1], which is well resolved from that of water, thus implying slow exchange of protons on the NMR timescale. We used the similarity of the concentrations of exchangeable protons of urea and water to measure more readily, than is typi- cally the case, the apparent rate constant that characterizes the exchange reaction. The corresponding 2-dimensional exchange spectra (2D-EXSY) were subjected to back-transformation analysis [2–5] to yield estimates of the apparent rate constant of the exchange reaction. The significance of these estimates of rate constants lies in defining the timescales of the de-protonation/re-protonation events that occur simultaneously with enzymic and membrane transport reactions. When the gel was held stretched [6–8], splitting of the amide resonance occurred as the result of intra-molecular residual dipo- lar couplings (RDCs); and with [1,3- 15 N]urea, the one-bond scalar coupling with the heteronuclei (| 1 J NH |  90 Hz) was added to the RDC splitting. The known sign of this coupling was used to inter- pret the sign of the RDC. When a mixture of 1 H 2 O and 2 H 2 O was used, the 1 H NMR spectrum of the amide protons in the stretched gels appeared as a doublet of triplets. We assigned this multiplet to mono- and di- protonated amides of urea and accounted for the relative intensi- ties of the peaks by considering the relative concentrations of all 16 possible isotopologues of protonated/deuterated urea, and their relative contributions to the intensities of the components of the split resonance. http://dx.doi.org/10.1016/j.jmr.2014.08.004 1090-7807/Ó 2014 Elsevier Inc. All rights reserved. Abbreviations: NMR, nuclear magnetic resonance; RDC, residual dipolar cou- pling; RQC, residual quadrupolar coupling; nOe, nuclear Overhauser effect. ⇑ Corresponding author. Address: School of Molecular Bioscience, Building G08, University of Sydney, NSW 2006, Australia. Fax: +61 2 9351 4726. E-mail address: philip.kuchel@sydney.edu.au (P.W. Kuchel). Journal of Magnetic Resonance 247 (2014) 72–80 Contents lists available at ScienceDirect Journal of Magnetic Resonance journal homepage: www.elsevier.com/locate/jmr