CHANGES IN APPARENT PH ON FREEZING AQUEOUS BUFFER SOLUTIONS AND THEIR RELEVANCE TO BIOCHEMICAL ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY
CHANGES IN APPARENT PH ON FREEZING AQUEOUS BUFFER SOLUTIONS AND THEIR RELEVANCE TO BIOCHEMICAL ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY
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DOI：
10.1042/bj1670593
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发表时间：
1977-01-01
影响因子：
4.1
通讯作者：
VINCENT, SP
中科院分区：
文献类型：
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作者：
WILLIAMSSMITH, DL;BRAY, RC;VINCENT, SP
在缓冲水溶液快速冷冻和冷却至-196度时发生的表观pH值变化。通过各种半定量方法研究C，包括简单的视觉测量pH指标的颜色变化，以及测量3种不同金属酶溶液的EPR光谱的pH依赖性变化。在特定条件下，可出现高达约3个pH单位的表观pH变化。这些变化与冷冻样品的时间无关，冷冻时间从3毫秒到20秒不等，但受到溶液中某些蛋白质的影响。建议在EPR光谱和其他低温生化工作中使用缓冲液以避免这种明显的pH变化。磷酸盐和焦磷酸盐缓冲液会使pH值大幅降低（2-3个pH单位），而Tris缓冲液在某些条件下会使pH值增加大约相同的幅度，因此应避免使用。某些两性离子缓冲液如比辛[nn -双-（2-羟乙基）甘氨酸]是令人满意的。发现表观pH效应取决于缓冲液和蛋白质浓度。作为今后详细的低温生化工作的前奏，应采用指标体系进行适当的试验。
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.