蛋白质与蛋白组学

3. Buffer Selection
There are many factors that must be considered when choosing a buffer. When studying an enzyme one must consider the pH optimum of the enzyme, nonspecific buffer effects on the enzyme, and interactions with substrates or metals. When purifying a protein, cost becomes an important consideration, as does the compatibility of the buffer with different purification techniques. Table 6.1 lists a wide variety of buffers covering a broad pH range.

缓冲液选择
缓冲液的选择有很多因素需要考虑。研究酶要考虑酶的最佳pH，非特异性缓冲液对酶的影响，及它对底物或金属的相互作用。纯化蛋白其成本是个着重考虑点，还要考虑缓冲液与不同纯化技术的相容性。表6.1列出了光谱pH范围的各种缓冲液。

Determining the pH optimum of a protein is a first step in determining the best buffer to employ (Blanchard, this series). Since the buffering capacity is maximal at the pK, buffers should be used close to this value. When determining the pH optimum for an enzyme, it is useful to use a series of related buffers that span a wide pH range. Once an optimal pH has been approximated, different buffers within this pH range can be examined for specific buffer effects. The Good buffers have been shown to be relatively free of side effects.

确定某个蛋白的最佳pH是决定选用最佳缓冲液的第一步。在pK值时，缓冲液的缓冲容量最大，所以应用的缓冲液要接近它的pK值。确定某个酶的最佳pH时，应用一系列pH宽范围的相关缓冲液是有用的。一旦估计出最佳pH，在此pH范围内的各种不同缓冲液就可以用来检测特异性的缓冲液影响。Good缓冲液一般不利影响相对较小。

惭愧，做了这么多年的蛋白纯化，竟然不知道"Good's" Buffers是什么东西。放狗搜，含义如下：
第一个紧密型的解释：
"Good's" Buffers refer to the group of buffers described in the research of Dr. Norman Good et al. in 1966. These buffers were selected because they display characteristics making them integral to research in biology and biochemistry. The characteristics associated with a Good's buffer include the following: pKa value between 6.0 and 8.0, high solubility, non toxic, limited effect on biochemical reactions, very low absorbence between 240 nm and 700 nm, enzymatic and hydrolytic stability, minimal changes due to temperature and concentration, limited effects due to ionic or salt composition of the solution, limited interaction with mineral cations, and limited permeability of biological membranes. (Reference: Good, N.E., et al. (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry 5(2), 467-477.)
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However, inorganic buffers do have a high potential for specific buffer effects. Many enzymes are inhibited by phosphate buffer, including carboxypeptidase, urease, as well as many kinases and dehydrogenases (Blanchard, this series). Borate buffers can form covalent complexes with mono- and oligosaccharides, the ribose moieties of nucleic acids, pyridine
nucleotides, and other gem-diols. Tris and other primary amine buffers may form Schiff base adducts with aldehydes and ketones.

但是，无机盐缓冲液具有很高潜质的特异性缓冲液作用。很多酶可以被磷酸盐缓冲液所抑制，包括羧肽酶、脲酶，很多激酶和脱氢酶。硼酸盐缓冲液可能形成共价复合物，如单糖、寡糖、核酸的核糖部分、吡啶、核苷酸和其他的二羟基化合物。Tris和其他基本氨基缓冲液可能与醛酮类形成Schiff碱加成反应物。

还好，这些这个酶那个酶的都与我无关，我还是用我的PB和Tris好了，呵呵。

Buffer complexation with metals may present additional problems. In this respect, inorganic buffers can prove problematic in that they may remove, by chelation, metals essential to enzymatic activity (e.g., Mg2t for kinases, Cu2t or Fe2t for hydroxylases). Release of protons upon chelation or precipitation of metal–buffer complexes may also be a potential problem. Where metal chelation presents a problem, the Good buffers are useful since they have been shown to have low metal-binding capabilities (Good et al., 1966). Once a suitable buffer has been found (noninteracting, with an appropriate pK), a concentration should be chosen. Since high ionic strength may decrease enzyme activity, the buffer concentration should be as low as possible (Blanchard, this series). A reasonable way to determine how low a concentration may be used is to examine the properties (reaction rate or protein stability) at a low (10–20 mM) concentration of buffer. The pH prior to, and an adequate time after, addition of protein should not vary more than _0.05 pH. If the pH changes too drastically (greater than _0.1 pH unit), then the buffer concentration should be raised to 50 mM. In cases where protons are consumed or released stoichiometrically with substrate utilization, pH stability becomes increasingly important.
（略）

几个注意点：
1、防止维持酶活性的金属离子(e.g., Mg2t for kinases, Cu2t or Fe2t for hydroxylases)被无机缓冲液所鳌合或去除（沉淀？）
2、Good buffers的金属离子鳌合性能低。对文章中的此点持保留。因为Good中有好些都有EDTA螯合剂，它怎么可能金属离子鳌合性能低呢？
3、尽量使用低浓度的缓冲液，如10-20mM。
4、如果酶反应消耗质子，pH的稳定性更为重要。

Buffers may be made up in stock solutions, then diluted for use. When stock solutions are made, it should be done close to the working temperature, and in glass bottles (plastic bottles can leach UV-absorbing material) (Perrin and Dempsey, 1974). Buffers have temperature-sensitive pK values, particularly amine buffers. The carboxylic acid buffers are generally the least sensitive to temperature, and the Good buffers have only a small inverse temperature dependence on pK. The effects of dilution of stock solutions, or addition of salts, on pH should be checked by measurement of the pH after addition of all components.

缓冲液可以配制成高浓度的储存液，使用时稀释。储液应该在接近工作温度的环境下的玻璃容器中配制（塑料瓶可能释放紫外吸收物质）。缓冲液的pK值有温度敏感性，特别是氨基缓冲液。羧酸缓冲液一般对温度不敏感，温度对Good缓冲液pK值仅有很小的影响。储存液的稀释，或盐的加入对pH的影响应该要校正，校正应该在所有组分加入后进行。

这一段很有意义。温度变化，母液的稀释，盐的加入，这些因素对缓冲液pH的影响要充分认识到。在蛋白质纯化过程中，这都是非常常见的现象。例如，Tris缓冲液在室温和4度的差别，镍柱纯化时咪唑的加入，离子交换柱纯化时氯化钠的加入都对缓冲液的pH有较大的影响。不注意的话，可能试验出了问题还不知道如何去找原因。一句话，纯化所用的缓冲液在最终配制完毕后最好用pH计检测一下pH是否正确！这对离子交换层析尤其重要。

Choosing a buffer for protein purification requires some special considerations. Large amounts of buffer will be needed for centrifugation, chromatographic separations, and dialysis, which makes cost a concern. Tris and many inorganic buffers are widely used since they are relatively inexpensive. Although buffers like Tris are inexpensive, and have been widely used in protein purification, they do have disadvantages. Tris is a poor buffer below pH 7.5 and its pK is temperature dependent (a solution made up to pH 8.06 at 25 C will have a pH of 8.85 at 0 C). Many primary amine buffers such as Tris and glycine (Bradford, 1976) will interfere with the Bradford dye-binding protein assay. Some of the Good buffers, HEPES, EPPS, and Bicine, give false-positive colors with Lowry assay.

蛋白质纯化用缓冲液的选择需要一些特别的考虑。离心、层析和透析都需要大量的缓冲液，成本要考虑到。Tris和很多无机缓冲液被广泛使用是由于它们相对便宜。尽管Tris之类的缓冲液便宜，并在蛋白质纯化中广泛使用，但我们不能忽视它们的缺点。Tris在pH7.5以下的缓冲容量很低，它的pK值温度依赖性很强（25度时的溶液pH8.06，在0度时为8.85）。很多基本的氨基缓冲液，如Tris和甘氨酸，会干扰Bradford法的蛋白分析。一些Good缓冲液，如HEPES, EPPS, and Bicine，会对Lowry法分析造成假阳性颜色。

氨基缓冲液会干扰Bradford，HEPES会干扰Lowry，要有印象，记住。

Spectroscopic measurement of enzyme rates is a commonly applied method. It may be important to use a buffer that does not absorb appreciably in the spectral region of interest. The Good buffers, and most buffers listed in Table 6.1, can be used above 240 nm.

酶催化速率的光度分析方法很常用。所以在应用的光谱范围内，缓冲液没有丝毫光吸收就显得很重要。Good缓冲液，和表6.1中所列出的多数缓冲液都能在240nm以上使用。

这个倒是不一定的。没有吸收自然是好，但有吸收也没有很大的问题。做个参照blanking，把本底值扣除就可以了，反正在做光度分析的时候是一定要做blanking的，不管你的buffer有没有光吸收，这是体系的要求。

再延伸一下，做酶动力学光度分析，对分光光度计要求有恒温水浴装置，在买设备的时候要注意这点。酶反应可是和温度关系很大啊。

4. Buffer Preparation
Once a suitable buffer has been chosen it must be dissolved and titrated to the desired pH. Before titrating a buffer solution, the pH meter must be calibrated. Calibration should be done using commercially available pH standards, bracketing the desired pH. If monovalent cations interfere, or are being investigated, then titration with tetramethylammonium hydroxide can be done to avoid mineral cations. Similarly, the substitution of the most commonly used counteranion, chloride, with other anions such as acetate, sulfate, or glutamate, may have significant effects on enzyme activity or protein–DNA interactions (Leirmo et al., 1987). Stock solutions should be made with quality water (deionized and double-distilled, preferably) and filtered through a sterile ultrafiltration system (0.22 um) to prevent bacterial or fungal growth, especially with solutions in the pH 6–8 range. To prevent heavy metals from interfering, EDTA (10–100 mM) may be added to chelate any contaminating metals.

缓冲液配制
选定缓冲液后，要溶解、调节到目标pH。在对缓冲液进行调节之前，pH计必须先做校正。要用商品化的pH标准品进行校正，标准品的pH范围要包括目标pH。如果单价阳离子有干扰，或被研究，pH调节可以用四甲基氢氧化铵以避免引入矿物阳离子。同理，最常用的相反的阴离子，氯离子，其替代物如乙酸盐、硫酸盐或谷氨酸盐可能对酶活性或蛋白质-DNA相互作用有严重的影响。缓冲储存液要用有质量的水来配制，最好是去离子水和双蒸水，还要经过一个灭菌的超滤系统（0.22 um）进行过滤，以防止细菌或真菌繁殖，特别是在pH6-8之间的缓冲液。为了防止重金属干扰，可以加入EDTA（10-100mM）来鳌合污染的金属离子。

几点感想：
1、pH计的校正。那种红、绿、蓝三色的标准pH缓冲液最好用，不要去买那种固体的国产试剂用容量瓶配制，不值得。pH计的日常维护也很重要。pH电极平时应处于正确的保存缓冲液中，不能干啊。
2、四甲基氢氧化铵从来没有用过，放狗。
3、水。蛋白质纯化操作去离子水是最低要求。
4、“灭菌的超滤系统（0.22 um）进行过滤”这句话属于表述错误，应该是指过滤系统，不是超滤系统。超滤截留0.22um还能叫超滤吗？一个大漏勺了。
5、“加入EDTA防止重金属干扰”说得不错，但要考虑具体应用情况。比如说前面刚刚说过的对金属离子依赖的酶活性。另外，文中10-100mM的使用浓度是不是太高了？一般使用5mM以下吧。

5. Volatile Buffers
In certain cases, it is necessary to remove a buffer quickly and completely. Volatile buffers make it possible to remove components that may interfere in subsequent procedures. Volatile buffers are useful in electrophoresis, ion-exchange chromatography, and digestion of proteins followed by separation of peptides or amino acids. Most of the volatile buffers (Table 6.2) are transparent in the lower UV range except for the buffers containing pyridine (Perrin and Dempsey, 1974). An important consideration is interference in amino acid analysis (i.e., reactions with ninhydrin). Most volatile buffers will not interfere with ninhydrin if the concentrations are not too high (e.g., triethanolamine less than 0.1 M does not interfere).


挥发性缓冲液
（略）

挥发性缓冲液在纯化方面的应用可能主要是冻干考虑。但是我自己从来没有做过相应的试验。总是对这个挥发性存有疑问。有挥发性没有问题，可以理解。但是处于缓冲溶液中的物质能挥发的那么干净吗？最简单的氨水，氨味要挥发干净不知需要多少天？