Measuring fast hydrogen exchange rates by NMR spectroscopy Fatiha Kateb a , Philippe Pelupessy a, * , Geoffrey Bodenhausen a,b a Ecole Normale Supe ´rieure, De ´partement de Chimie, associe ´ au CNRS, 24 rue Lhomond, 75231 Paris Cedex 05, France b Ecole Polytechnique Fe ´de ´rale de Lausanne, Laboratoire de Re ´sonance Magne ´tique Biomole ´culaire, Batochime, CH-1015 Lausanne, Switzerland Received 31 July 2006; revised 12 September 2006 Available online 17 October 2006 Abstract We introduce a method to measure hydrogen exchange rates based on the observation of the coherence of a neighboring spin S such as 15 N that has a scalar coupling J IS to the exchanging proton I. The decay of S x coherence under a Carr–Purcell–Meiboom–Gill (CPMG) multiple echo train is recorded in the presence and absence of proton decoupling. This method allows one to extract proton exchange rates up to 10 5 s 1 . We could extend the pH range for the study of the indole proton in tryptophan, allowing the determination of the exchange constants of the cationic, zwitterionic, and anionic forms of tryptophan. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Hydrogen exchange; Proton transfer; Kinetic rates; Scalar relaxation Nuclear magnetic resonance is a powerful tool to inves- tigate hydrogen exchange [1]. Slow exchange rates can be measured by following in real time the replacement of hydrogen by deuterium [2]. Several methods have been developed to study fast and intermediate exchange, such as the analysis of the line-shape of exchanging protons [3], polarization transfer from the water resonance [4], decorrelation of longitudinal two-spin order [5] or mea- surements of translational diffusion coefficients [6]. Under favorable conditions, these methods allow one to measure rates up to a few thousand s 1 . It is also possible to inves- tigate the line-shape of a coupled nucleus, which is affected by scalar relaxation of the second kind [7]. For small exchange rates, the spectra feature doublets separated by the scalar coupling constant J IS , while in the limit of fast exchange the doublet collapses to a narrow singlet by a process called self-decoupling. Neglecting cross-correlated relaxation effects, the evolution of an initial in-phase coher- ence of a spin S such as 15 N coupled to an exchanging pro- ton 1 H with spin I can be obtained by solving the Liouville equation S x ðtÞ¼ S x ð0Þ expfvt  RðS x ÞtgfcoshðutÞ þðv=uÞ sinhðutÞg ð1Þ where v = k/2 and u ¼ðv 2  p 2 J 2 IS Þ 1=2 , k = k ex + R(2S x I z )  R(S x ), R(S x ) and R(2S x I z ) are the auto-relaxa- tion rates of S x and 2S x I z , while k ex is the exchange rate. In order to extract k, one can compare spectra recorded with and without proton decoupling [8]. However, in this case, long-range couplings n J I 0 S with n > 1 will also contrib- ute to the undecoupled line-width, which can lead to erro- neous measurements of fast exchange rates close to the self- decoupling limit. In order to eliminate these long-range couplings we propose to use a Carr–Purcell–Meiboom–Gill (CPMG) pulse-train [9] applied to the S spins and to detect the decay of the S x coherence with and without proton decoupling. The p pulses in this echo train need not be closely spaced: typically, an interval 2s = 10 ms between the pulses suffices to efficiently eliminate the effect of long-range scalar couplings n J I 0 S < 10 Hz, while the larger short-range couplings retain their efficiency as a vehicle of scalar relaxation. Let A be a signal amplitude propor- tional to the in-phase coherence S x remaining after a CPMG pulse-train without proton decoupling, and B a similar signal with decoupling. Fig. 1 shows the ratio A/B as a function of k for a scalar coupling J IS = 98.6 Hz, a 1090-7807/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jmr.2006.09.022 * Corresponding author. Fax: +33 1 44 32 33 97. E-mail address: philippe.pelupessy@ens.fr (P. Pelupessy). www.elsevier.com/locate/jmr Journal of Magnetic Resonance 184 (2007) 108–113