Changes in apparent pH on freezing aqueous buffer solutions and their relevance to biochemical electron-paramagnetic-resonance spectroscopy

Abstract

Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196.degree. C were studied by various semiquantitative methods, including simple visual measurements of color changes with pH indicators, as well as measurements of pH-dependent changes in the EPR spectra of solutions of 3 different metalloenzymes. 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 20 s, 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 EPR 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. As a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.