Research paper Precise in situ photo-induced pH modulation during NMR spectrometry Simli Dey a , Pavlo Bielytskyi b , Daniel Gräsing b , Anirban Das a , Rajasree Kundu a , Jörg Matysik b , Sudipta Maiti a,⇑ , P.K. Madhu c,⇑ a Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India b Institut für Analytische Chemie, Universität Leipzig, Linnéstraße 3, 04103 Leipzig, Germany c TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad 500107, India article info Article history: Received 13 May 2018 In final form 4 July 2018 Available online 9 July 2018 abstract We demonstrate a technique which can change the pH inside an NMR tube in steps of 0.01 pH unit, dur- ing a solution-state NMR experiment. We use a photo-acid co-solute with the actual specimen, and photo-excite the solution by direct fibre-mediated light delivery to the NMR tube. The photo- excitation and NMR measurements can be performed simultaneously. Our method should allow precise variation of the pH in studying protein folding, aggregation and enzyme catalysis processes which often display critical pH dependence. Ó 2018 Elsevier B.V. All rights reserved. 1. Introduction pH is a ubiquitous modulator of protein function, and serves as a very useful tool to study properties of proteins. In many enzyme- catalysed reactions, the catalytic centre consists of residues which can act as a proton donor or acceptor. Precise determination of the pKa of the relevant residues at the active site is therefore one of the keys to chemical reactivity [1–3]. The process of protein folding also depends critically on the pH for most proteins [4–8]. Similarly, protein aggregation is typically controlled by pH [9,10]. The con- ventional approach for studying these changes involves separate experiments on samples buffered at different pH values. The preci- sion and ease of performing these measurements can be consider- ably improved if the pH could be changed at will during the experiment, so that the same sample can be used for the whole pH dependent experiment. If the pH can be recycled up or down, then an additional benefit can accrue for experiments which probe the structure of the transient intermediates (e.g. in protein aggre- gation studies). If the pH is changed to a value where aggregation gets initiated, then one has only a finite window of time to mea- sure the structure of the intermediates. On the other hand, if the pH is periodically brought back to a value where the protein is sol- uble, then arbitrarily long measurements of an intermediate aggre- gation state can be performed until adequate signal to noise has been obtained. All of these have motivated us to develop a tech- nique which can in principle perform such pH modulation. Our idea is to have a pH changing agent as a co-solute with the speci- men of interest in the NMR tube, which can be controlled by an external agent during an NMR experiment. We decided to use a photo-acid, and direct in-tube light excitation through an optical fibre bundle [11,12], to release protons from the photo-acid and modulate the pH of the solution. If the light is turned on, the photo-acid will keep ejecting protons into the solution, thereby continuously lowering the pH (as long as the pH remains above the excited state pKa of the photo-acid). The rate of this lowering is completely dependent on the light intensity, which can be con- trolled at will, and the pH lowering can be stopped by turning the light off. In addition to the photoacid, we also included a pH repor- ter molecule in the solution. The reporter has a pH sensitive peak in the region of interest and reports the actual pH of the solution at any given time. The photo-excitation and NMR measurements do not interfere with each other and can be performed simultane- ously. With this approach, we demonstrate high accuracy in achieving any target pH in the near-physiological range during a solution NMR experiment. 2. Material and methods 2.1. Materials Imidazole (pH calibrant) and o-Nitrobenzaldehyde (o-NBA, pho- toacid) with 99% purity were purchased from Qualigens and S.D. Fine-Chem Ltd. (India) respectively. Deuterium Oxide with 99.9% D purity and Trimethylsilylpropanoic acid were obtained from Sigma Aldrich. https://doi.org/10.1016/j.cplett.2018.07.009 0009-2614/Ó 2018 Elsevier B.V. All rights reserved. ⇑ Corresponding authors. E-mail addresses: maiti@tifr.res.in (S. Maiti), madhu@tifr.res.in (P.K. Madhu). Chemical Physics Letters 706 (2018) 665–668 Contents lists available at ScienceDirect Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett