Changes in apparent pH on freezing aqueous buffer solutions and ...
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Changes in apparent pH on freezing aqueous buffer solutions and ...
Apr 6, 1977 - Department ofPhysics, Guy's Hospital Medical School, London SE1 9RT, U.K. ... pH changes during the freezing of buffer solutions containing ...
Biochem. J. (1977) 167, 593-600
593
Printed in Great Britain
Changes in Apparent pH on Freezing Aqueous Buffer Solutions and their Relevance to Biochemical Electron-Paramagnetic-Resonance Spectroscopy By DAVID L. WILLIAMS-SMITH Department ofPhysics, Guy's Hospital Medical School, London SE1 9RT, U.K. and ROBERT C. BRAY, MICHAEL J. BARBER, ALEXANDER D. TSOPANAKIS and STEPHEN P. VINCENT School of Molecular Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, U.K.
(Received 6 April 1977) Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196°C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3 pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3 ms to 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed. Investigations of biological molecules by physical methods are frequently carried out at low temperatures in frozen aqueous solutions (for early studies see, e.g., Keilin & Hartree, 1949). Such work, which may be performed at temperatures
