A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures

Abstract

A temperature independent pH buffer has been develeloped from combination of buffers of oppositesign temperature coefficients, and utility in low temperature spectroscopy and storage of pH sensitive compounds is demonstrated.Storage and analysis of samples at low and cryogenic temperatures has become a routine practice in modern research, as these temperatures can preserve integrity of precious samples, and allow modern biophysical and bioanalytical techniques to provide information on biomolecules at an unprecedented level. [1][2][3][4][5][6][7] Buffers are invariably used in the sample storage and analysis process. It is widely known that the pH of a buffer solution can change at low temperatures, and this has been ascribed to enthalpic effects on the proton equilibrium as well as selective precipitation of buffer components upon cooling. [8][9][10][11] If left unaccounted for, these pH changes could lead to demage to the samples and erroneous conclusions about biomolecular structures and dynamics at physiological temperature.About 75 years ago, Finn and coworkers reported the denaturation of proteins contained in muscle juice due to the variation in hydrogen ion and salt concentrations upon freezing. 12 Thereafter, activity loss of aldolase, phosphofructokinase and several dehydrogenases in sodium and potassium phosphate buffer at lower temperatures has been observed and ascribed to pH effects. 13,14 Several strategies have been applied to measure the temperature-dependent pH characteristics, such as the measurement of electromotive force (emf) to determine temperature dependence of pK a values, or the use of pH-sensitive dyes to probe protonic activity at low temperatures. [15][16][17][18] It was reported that EPR (electron paramagnetic resonance) based estimates of apparent pH change from the observation of several pH-sensitive systems (such as the flavin adenine dinucleotide semiquinone radical (FADH•) in xanthine oxidase) coincide well with estimates based on indicator dye optical changes. 19 Despite broad awareness, and extensive research into the relevance and merits of cryotemperature studies on biological systems, 20, 21 no reports of strategies to obtain buffers that resist temperature-dependent pH changes have appeared in the literature. Here we address this long standing problem through combination of buffers exhibiting an increase in pH upon freezing with those exhibiting a decrease. The utility of these temperature-independent-pH (TIP) buffers in preserving the sample integrity upon freezing of pH-sensitive pharmaceutical drugs, and in spectroscopic studies of human methemoglobin (met-Hb) is also demonstrated. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTo obtain the TIP buffer, the apparent pH changes of the two commonly used buffers, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and potassium phosphate) and mixtures thereof were first measured at low temperature via ratiometric absorption spectroscopy. A 1:1 mixture of two pH-indicator dyes (b...

Shift of pH-Value During Thermal Treatments in Buffer Solutions and Selected Foods

“…In view of the dissociation equilibrium shift during heating, a biased error in the results of inactivation experiments occurs, which leads to incomparable results if they are not designed correctly. Several authors have shown that the thermal inactivation of bacterial spores, [8][9][10][11][12] vegetative microorganisms, [13] or protein and enzyme denaturation [14][15][16] strongly depends on the pH of the suspending media. According to Goldberg, [17] the known temperature dependence of the acid dissociation constant K a (T) for different buffer systems could be interpolated up to 60 • C.…”

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

“…In general, an increase of pH by 0.6 – 1.1 units has been detected at low temperatures. To avoid such changes, a new method was developed recently (46), which allows to create buffers with temperature-independent pH by mixing of two buffers, one with positive and another with negative temperature coefficients of their pH. This approach can be very useful if constant pH has to be maintained in the broad temperature range.…”

Biological pH buffers in IVF: help or hindrance to success

“…Using spectrophotometric methods, it has been observed that the apparent pH of phosphate buffered solutions decreases significantly upon slow rate freezing [48]. Similarly, Quinn noted that in examination of the color of a phosphate buffered medium with phenol red used during slow rate freezing, at −80°C, the "color is yellow", indicating a severe acidification [66] (Fig.…”

“…At least one study has attempted to address this issue of temperature and change in pHe of buffered media by formulating a temperature independent pH (TIP) buffer [48]. By utilizing spectroscopic assessment of color change of a pH indicator included the media, researchers combined two buffers, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) and a phosphate-buffered solution.…”