Tryptophan proton exchange

NH resonances often appear in a spectrum when the substance is dissolved in H2O instead of H20. Proton exchange is then slow, especially when it is hydrogen-bonded or lies in a pocket inaccessible to bulk solvent. Hydrogen-bonded and tryptophan-NH protons can usually be located to low field, conveniently several ppm outside the main spectral envelope. 

Five of the 6 tryptophan-NH resonances of lysozyme (mol. wt. 14400) in H2O are found at 10-12 ppm from DSS [33]. These protons take several hours to exchange with H when dissolved in H20. Their differential exchange rates and response to substrates are an aid to assignment. Eleven protons of bovine pancreatic trypsin inhibitor take more than 4 months to exchange with H in H20 at pH 7 [34,35]. Evidently some domains of this small protein (58 amino acids) are extremely tightly folded. 

The indole singlet excited state can be quenched by proton transfer to the indole, and this can lead to deuterium exchange of the ring hydrogens [10]. In the case of tryptophan, the conjugate acid of the side-chain amine can act as the proton source [11], and an intermediate assigned as the 4-protonated indole 3 has been observed by flash photolysis [12]. 

The resonances of the indole NH protons for five of the six tryptophan residues of hen egg-white lysozyme were resolved by observing the NMR spectrum in water 147). Resonances were assigned to specific tryptophan residues by differential deuterium exchange rates, inhibitor perturbation and chemical modification. Well resolved indole NH resonances were obtained for all five tryptophan residues of human lysozyme. 

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