【文章內(nèi)容簡(jiǎn)介】 s selectively catalyze these cell wall reactions, they react only sparingly with other solutes within the cell. 酶消化法的缺點(diǎn)在于酶的價(jià)格昂貴限制了在大規(guī)模生產(chǎn)中的使用 ，雖然條件溫和 、 具有選擇性 。 細(xì)胞懸浮液中加入酶能迅速和細(xì)胞壁反應(yīng)并破壞它們 。 酶選擇性的催化細(xì)胞壁反應(yīng) ， 不破壞細(xì)胞內(nèi)的其它物質(zhì) 。 Alkali treatment is the antithesis of enzyme digestion, for it is harsh rather than gentle, nonselective rather than specific, and cheap rather than expensive. Alkali added to a cell suspension reacts with the cell walls in a number of ways, including the saponification of lipids in the cell walls. 堿處理法和酶消化法相反，反應(yīng)激烈，不具選擇性，而且較便宜。堿加入細(xì)胞懸浮液中后和細(xì)胞壁進(jìn)行了多種反應(yīng)，包括使磷脂皂化。 In a sense, the treatment is an extreme example of so1ubilization, for it converts cell wall ponents into detergents. Including protein denaturations. As a result, the method tends both to rupture cell walls and to destroy products. Even though it is cheap, alkali treatment is less useful than the three methods which are detailed next. 該操作是細(xì)胞增溶的很好的例子 ， 因?yàn)樗辜?xì)胞壁的成分溶于表面活性劑 ， 也使蛋白質(zhì)變性 。 該法不僅破壞了細(xì)胞壁也破壞了產(chǎn)物 ， 因此即便很便宜 ， 堿處理也是一種很不常用的方法 。 A、 Osmotic Shock(滲透沖擊法 ) The simplest of the three major chemical methods is osmotic shock. This is nothing more than dumping a given volume of cells into pure water often about twice the volume of cells. The cells swell because they contain solutes which cause an osmotic flow of water into the cells. In some cases, they swell so much that they burst. Their contents, released into the surrounding solution, can now be separated using the methods in following chapters. 三種主要細(xì)胞破碎化學(xué)方法中最簡(jiǎn)單的是滲透沖擊法。此法將一定體積的細(xì)胞液加到 2倍體積的水中，細(xì)胞中溶質(zhì)濃度高，水不斷進(jìn)入細(xì)胞，使細(xì)胞膨脹，最后導(dǎo)致破裂。細(xì)胞破裂后釋放到周圍環(huán)境中的胞內(nèi)物可用后面章節(jié)介紹的方法分離。 The susceptibility of cells to lysis depends strongly on their type. Red blood cells are easily lysed. Animal cells often can be lysed, but only after animal tissue has been mechanically minced or homogenized, as described in Section . Plant cells are much more difficult to 1yse, for their cell walls often contain strong woody material which is relatively impermeable to osmotic flow. 細(xì)胞破碎的難易決定于其類型，紅血球細(xì)胞容易溶破，動(dòng)物細(xì)胞只有當(dāng)其組織被用 。植物細(xì)胞很難溶破，因?yàn)橹参锛?xì)胞中含有大量的木質(zhì)成分，通過滲透流很難滲透。 The osmotic flow involved es from the osmotic pressure, which is surprisingly large. To estimate the size of this pressure, we turn to the definition of chemical equilibrium, which states that the chemical potential of water 181。H20 must be constant： 滲透流的動(dòng)力來自滲透壓，滲透壓可能很大?？梢酝ㄟ^化學(xué)平衡來估算，水的化學(xué)電勢(shì)是常數(shù)，即： 181。H20 (outside) = 181。H20 (inside) () The chemical potential of the pure water outside must include a reference value and a pressure correction。 the corresponding potential inside involves a reference value, pressure correction, and a correction for solution concentration. For an ideal inpressible solution, these corrections convert Eq. () into the following. 細(xì)胞外純水的化學(xué)電勢(shì)包括標(biāo)準(zhǔn)化學(xué)電勢(shì)和壓力修正項(xiàng)；與之平衡的胞內(nèi)的化學(xué)電勢(shì)包括三項(xiàng)，即標(biāo)準(zhǔn)化學(xué)電勢(shì)、壓力修正相和濃度修正項(xiàng)。對(duì)于理想的不可壓縮溶液，這些修正相和式（ ）可改寫為： 181。0 H20+VH2oPout = 181。0 H20+VH2oPin+RT ln(1x1) () Where 181。0H20 is the reference value,VH2o is the partial molar volume of water, and x1 is the total mole fraction of solutes inside the cell. If the cell’s contents are a dilute solution, VH2o is almost equal to the molar volume of water VH2o , and x1 is small 181。0H20—— 標(biāo)準(zhǔn)化學(xué)電勢(shì) VH2o—— 水的偏摩爾體積 x1—— 細(xì)胞內(nèi)所有溶質(zhì)的總摩爾分?jǐn)?shù) 若包內(nèi)物為稀溶液，其偏摩爾體積等于純水的摩爾體積。且 x1很小時(shí)就有： Thus PoutPin=(RT/VH2o) ln(1x1) =(RT/VH2o) (x1…) =RTc1+ … ( 3) This relation is called van’t Hoff’s law.(該式稱為范特苛夫定律。 ) Thus at equilibrium, the pressure outside the cell must be less than that inside. If this is not the case, then water will flow into the cell and potentially lyses the cell. We can use Eq. () to estimate the size of the pressure. Many cells have a solute concentration roughly equivalent to M NaCl, or M solutes. 由該方程知細(xì)胞內(nèi)壓力必須小于細(xì)胞外壓力，否者，水會(huì)流入細(xì)胞內(nèi)，溶破細(xì)胞。我們可以采用等式 估算出滲透壓的大小。許多細(xì)胞內(nèi)溶質(zhì)濃度大約為 NaCl 或 。 This osmotic pressure is, for us, always surprisingly large. This large pressure is what can lead to cell rupture. 由此可見滲透壓必須很大，才導(dǎo)致了細(xì)胞的破碎。 B 、 Solubilization 增溶法 The second major method of chemically rupturing cells is solubilization by detergents. Typically,a concentrated detergent solution is added to about half the solution’s volume of cells. The detergent disrupts the cell membrane. The resulting suspension can be centrifuged to remove cell fragments, and then run through an adsorption column or an extractor to isolate the product. 第二種是利用表面活性劑的增溶法。最典型的是將體積為細(xì)胞體積兩倍的某濃度的表面活性劑加入到細(xì)胞中。表面活性劑能將細(xì)胞壁破碎，制成的懸浮液可用離心分離除去細(xì)胞碎片，再用吸附柱或萃取劑分離制得產(chǎn)品。 The reason that solubilization is effective lies in detergent chemistry. This chemistry depends on detergent structures like those in Figure . All these structures have a hydrophilic portion, which is often ionic, and a hydrophobic part, which is frequently a hydrocarbon. 方法有效在于表面活性劑的化學(xué)性質(zhì)，化學(xué)性質(zhì)由圖 化學(xué)結(jié)構(gòu)表示。結(jié)構(gòu)中有一個(gè)親水基團(tuán)，通常是離子；一個(gè)疏水基團(tuán)，通常是烴基。 As a result, these detergents are all amphipathic, capable of interacting with both water and lipid. 表面活性劑通常是兩性的，既能和水作用也能和脂作用。 This amphipathic nature holds whether the detergents are anionic, cationic, or nonionic. All types are illustrated in the figure. Sodium dodecylsulfate (