Early fractionation steps in a purification utilize differences in protein solubility, which is a complex function of pH, temperature, salt concentration, and other factors. The solubility of proteins is lowered in the presence of some salts, an effect called “salting out.” Ammonium sulfate ((NH4)2SO4) is particularly effective for selectively precipitating some proteins while leaving others in solution. Low-speed centrifugation is then used to remove the precipitated proteins from those remaining in solution.

纯化中早期的分级分离步骤利用的是蛋白质溶解度的差异，而溶解度是 pH、温度、盐浓度以及其他因素的复杂函数。某些盐存在时蛋白质的溶解度下降，这一效应称为「盐析」。硫酸铵 ((NH₄)₂SO₄) 特别有效，可以选择性地沉淀一部分蛋白质而让另一些留在溶液中。随后用低速离心把沉淀下来的蛋白质与仍留在溶液中的蛋白质分开。

Ionic interactions between dissolved ions are much stronger in less polar environments, because there is less screening of charges by the nonpolar solvent. Water is effective in screening the electrostatic interactions between dissolved ions because it has a high dielectric constant, a physical property that reflects the number of dipoles in a solvent.

在极性较低的环境中，溶解离子之间的离子相互作用要强得多，因为非极性溶剂对电荷的屏蔽较弱。水之所以能有效屏蔽溶解离子之间的静电相互作用，是因为它具有高介电常数——这一物理性质反映了溶剂中偶极子的数目。

Proteins can also be denatured by extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or by detergents. Each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken. Organic solvents, urea, and detergents act primarily by disrupting the hydrophobic aggregation of nonpolar amino acid side chains that produces the stable core of globular proteins; urea also disrupts hydrogen bonds; and extremes of pH alter the net charge on a protein, causing electrostatic repulsion and the disruption of some hydrogen bonding.

蛋白质还可以被极端 pH、某些可与水混溶的有机溶剂（如乙醇或丙酮）、某些溶质（如尿素和盐酸胍）或去污剂所变性。这些变性剂中的每一种都属于相对温和的处理，因为多肽链中并没有共价键被破坏。有机溶剂、尿素和去污剂主要通过破坏非极性氨基酸侧链的疏水聚集（正是这种聚集形成了球状蛋白稳定的内核）而起作用；尿素还会破坏氢键；极端 pH 则改变蛋白质的净电荷，引起静电排斥并破坏部分氢键。

Denaturation often leads to protein precipitation, a consequence of protein aggregate formation as exposed hydrophobic surfaces associate. The aggregates are often highly disordered. One example is the protein precipitate that can be seen after an egg white is boiled. More-ordered aggregates are also observed in some proteins, as we shall see.

变性常常导致蛋白质沉淀，这是暴露出来的疏水表面彼此结合、形成蛋白质聚集体的后果。这些聚集体往往高度无序。一个例子就是煮沸蛋清后可以看到的蛋白质沉淀。在某些蛋白质中也能观察到较为有序的聚集体，后文会讲到。

polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In electrophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility, μ, of a molecule is the ratio of its velocity, V, to the electrical potential. Electrophoretic mobility is also equal to the net charge, Z, of the molecule divided by the frictional coefficient, f, which reflects in part a protein’s shape.

（聚丙烯酰胺）凝胶起分子筛的作用，按蛋白质的荷质比大致成比例地减慢其迁移。迁移也会受到蛋白质形状的影响。在电泳中，驱动大分子移动的力是电势 E。分子的电泳迁移率 μ 是其速度 V 与电势之比。电泳迁移率也等于分子的净电荷 Z 除以摩擦系数 f，而摩擦系数部分地反映了蛋白质的形状。

of the bound SDS contribute a large net negative charge, rendering the intrinsic charge of the protein insignificant and conferring on each protein a similar charge-to-mass ratio. In addition, SDS binding partially unfolds proteins, such that most SDS-bound proteins assume a similar rodlike shape. Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypeptides migrating more rapidly. After electrophoresis, the proteins are visualized by adding a dye such as Coomassie blue, which binds to proteins but not to the gel itself (Fig. 3-18b). Thus, a researcher can monitor the progress of a protein purification procedure as the number of protein bands visible on the gel decreases after each new fractionation step. When compared with the positions to which proteins of known molecular weight migrate in the gel, the position of an unidentified protein can provide a good approximation of its molecular weight (Fig. 3-19). If the protein has two or more different subunits, generally the subunits are separated by the SDS treatment, and a separate band appears for each.

（结合上的 SDS 的硫酸基团）带来很大的净负电荷，使蛋白质自身固有的电荷变得无足轻重，并赋予每种蛋白质相近的荷质比。此外，SDS 的结合会使蛋白质部分去折叠，因而大多数结合了 SDS 的蛋白质呈现出相似的棒状形态。因此，在 SDS 存在下的电泳几乎完全按质量（分子量）分离蛋白质，分子越小的多肽迁移越快。电泳之后，用考马斯亮蓝一类的染料使蛋白质显现出来，这种染料结合蛋白质而不结合凝胶本身（图 3-18b）。这样，随着每一步新的分级分离之后凝胶上可见蛋白质条带数目的减少，研究者就能监测蛋白质纯化的进程。把未知蛋白质在凝胶中的位置与已知分子量的蛋白质所迁移到的位置相比较，就能很好地估计出它的分子量（图 3-19）。如果蛋白质含有两个或更多不同的亚基，一般来说这些亚基会被 SDS 处理解离开，每一个亚基各出现一条带。

In one application of this technique, an immunoblot or Western blot assay (Fig. 5-26b), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. After washing, the membrane is treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms only along the band containing the protein of interest. Immunoblotting allows the detection of a minor component in a sample and provides an approximation of its molecular weight.

这一技术的一个应用是免疫印迹（immunoblot）或 Western blot 分析（图 5-26b）：经凝胶电泳分离的蛋白质被电转移到硝酸纤维素膜上。洗涤之后，膜依次用一抗、连有酶的二抗以及底物处理。只有在含目的蛋白质的那条带处才会形成有色沉淀。免疫印迹既能检出样品中的微量组分，又能给出它的近似分子量。

This technique separates proteins according to their isoelectric points. A protein mixture is placed on a gel strip containing an immobilized pH gradient. With an applied electric field, proteins enter the gel and migrate until each reaches a pH that is equivalent to its pI. Remember that when pH = pI, the net charge of a protein is zero.

这一技术按蛋白质的等电点把它们分离开。把蛋白质混合物加到含有固相化 pH 梯度的胶条上。施加电场后，蛋白质进入凝胶并迁移，直到各自到达与其 pI 相等的 pH 处为止。记住：当 pH = pI 时，蛋白质的净电荷为零。

Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional electrophoresis permits the resolution of complex mixtures of proteins (Fig. 321). This is a more sensitive analytical method than either electrophoretic method alone.

把等电聚焦与 SDS 电泳依次结合起来，这一过程称为双向电泳，它能够分辨复杂的蛋白质混合物（图 3-21）。这是一种比单用其中任何一种电泳方法都更灵敏的分析方法。

Proteins are first separated by isoelectric focusing in a thin strip gel. The gel is then laid horizontally on a second, slab-shaped gel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. Horizontal separation reflects differences in pI; vertical separation reflects differences in molecular weight. The original protein complement is thus spread in two dimensions. Thousands of cellular proteins can be resolved using this technique. Individual protein spots can be cut out of the gel and identified by mass spectrometry (see Figs 3-28 and 3-29).

蛋白质先在一条薄胶条中通过等电聚焦分离。然后把该胶条水平铺放在第二块片状凝胶上，用 SDS 聚丙烯酰胺凝胶电泳分离蛋白质。水平方向的分离反映 pI 的差别，垂直方向的分离反映分子量的差别。原有的全部蛋白质就这样在二维平面上被展开。用这一技术可以分辨数千种细胞蛋白质。单个蛋白质斑点可以从凝胶上切下来，用质谱加以鉴定（见图 3-28 和 3-29）。

Unknown purified proteins can be identified, and their mass can be accurately determined. When coupled to powerful peptide separation protocols, mass spectrometry can document a complete cellular proteome —defined as the entire complement of proteins in a cell, including estimates of their relative abundance — in just an hour.

未知的纯化蛋白质可以被鉴定，其质量也能被精确测定。当与强有力的肽段分离方案联用时，质谱只需一小时就能记录下一个完整的细胞蛋白质组——蛋白质组定义为细胞内全部蛋白质的总和，其中还包括对它们相对丰度的估计。

Tryptophan and tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet light (Fig. 36; see also Box 3-1). This accounts for the characteristic strong absorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the characterization of proteins.

色氨酸和酪氨酸——以及程度小得多的苯丙氨酸——能吸收紫外光（图 3-6；另见框 3-1）。这解释了大多数蛋白质在 280 nm 波长处特征性的强光吸收，研究者正是利用这一性质来表征蛋白质。

A wide range of biomolecules absorb light at characteristic wavelengths, just as tryptophan absorbs light at 280 nm (see Fig. 3-6). Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution. The fraction of the incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species (Fig. 1).

多种多样的生物分子都在各自特征波长处吸收光，正如色氨酸在 280 nm 处吸光一样（见图 3-6）。用分光光度计测量光吸收，可用于检测和鉴定分子，并测定它们在溶液中的浓度。溶液在给定波长下吸收入射光的比例，与吸光层的厚度（光程）以及吸光物质的浓度有关。

By international agreement, 1.0 unit of enzyme activity for most enzymes is defined as the amount of enzyme causing transformation of 1.0 μmol of substrate to product per minute at 25 °C under optimal conditions of measurement (for many enzymes, this definition is inconvenient, and a unit may be defined differently). The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3-22). The specific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3-5).

按国际约定，对大多数酶而言，1.0 个酶活力单位定义为在最适测定条件下、25 °C 时每分钟使 1.0 μmol 底物转变为产物所需的酶量（对许多酶来说这一定义并不方便，单位可以另行定义）。「活力」一词指溶液中酶的总单位数。「比活力」是每毫克总蛋白所含的酶单位数（图 3-22）。比活力是酶纯度的量度：在酶的纯化过程中比活力不断升高，当酶纯净时达到最大并保持恒定。

For proteins that are not enzymes, other quantification methods are required. Transport proteins can be assayed by their binding to the molecule they transport, and hormones and toxins by the biological effect they produce; for example, growth hormones will stimulate the growth of certain cultured cells. Some structural proteins represent such a large fraction of a tissue mass that they can be readily extracted and purified without a functional assay. The approaches are as varied as the proteins themselves.

对于不是酶的蛋白质，需要其他的定量方法。转运蛋白可以通过它与所转运分子的结合来测定，激素和毒素则通过它们产生的生物学效应来测定，例如生长激素会刺激某些培养细胞的生长。有些结构蛋白在组织质量中所占比例很大，不需要功能测定就能容易地提取和纯化。这些方法与蛋白质本身一样多种多样。

By fusing the gene encoding a protein under study with the gene for an epitope tag, investigators can precipitate the protein product of the fusion gene by complexing it with the antibody that binds the epitope. This process is called immunoprecipitation (Fig. 9-18). If the tagged protein is expressed in cells, other proteins that bind to it precipitate with it. Identifying the associated proteins reveals some of the intracellular protein-protein interactions of the tagged protein.

把编码待研究蛋白质的基因与编码表位标签的基因融合，研究者就能通过让融合基因的蛋白质产物与结合该表位的抗体形成复合物，把它沉淀下来。这一过程称为免疫沉淀（图 9-18）。如果带标签的蛋白质在细胞中表达，那么与之结合的其他蛋白质也会随之一起沉淀。鉴定这些相伴沉淀的蛋白质，就揭示出带标签蛋白质在细胞内的部分蛋白质-蛋白质相互作用。

The selectivity of this approach can be enhanced with tandem affinity purification (TAP) tags. Two consecutive tags are fused to a target protein, and the fusion protein is expressed in a cell (Fig. 9-19). The first tag is protein A, a protein found at the surface of the bacterium Staphylococcus aureus that binds tightly to mammalian immunoglobulin G (IgG). The second tag is often a calmodulin-binding peptide.

这一方法的选择性可以用串联亲和纯化（TAP）标签来提高。把两个连续的标签融合到目标蛋白质上，并让融合蛋白在细胞中表达（图 9-19）。第一个标签是蛋白 A，它是金黄色葡萄球菌表面的一种蛋白质，能与哺乳动物免疫球蛋白 G（IgG）紧密结合。第二个标签常常是钙调蛋白结合肽。

In yeast two-hybrid analysis, the protein-coding regions of the genes to be analyzed are fused to the yeast gene for either the DNA-binding domain or the activation domain of Gal4p, and the resulting genes express a series of fusion proteins (Fig. 9-20). If a protein fused to the DNA-binding domain interacts with a protein fused to the activation domain, transcription is activated. The reporter gene transcribed by this activation is generally one that yields a protein required for growth or an enzyme that catalyzes a reaction with a colored product.

在酵母双杂交分析中，把待分析基因的蛋白质编码区分别与酵母 Gal4p 的 DNA 结合结构域基因或转录激活结构域基因融合，所得基因表达出一系列融合蛋白（图 9-20）。如果融合到 DNA 结合结构域上的蛋白质与融合到激活结构域上的蛋白质发生相互作用，转录就被激活。由这种激活所转录的报道基因，一般产生一种生长所必需的蛋白质，或者一种催化生成有色产物的反应的酶。

How do we know that two proteins interact, or where in the cell they interact? Changes in the state of association of two proteins (such as the R and C subunits of PKA) can be seen by measuring the nonradiative transfer of energy between fluorescent probes attached to each protein, a technique called fluorescence resonance energy transfer (FRET), which we describe in Box 12-1.

我们怎样知道两个蛋白质发生了相互作用，或者它们在细胞的什么部位相互作用？两个蛋白质（例如 PKA 的 R 亚基和 C 亚基）缔合状态的改变，可以通过测量连接在各自蛋白质上的荧光探针之间的非辐射能量转移来观察，这一技术称为荧光共振能量转移（FRET），我们在框 12-1 中加以介绍。