Biochemistry - Chemical Reactions in Living Cells, Volume 1 - D. Metzler 1980

The Molecules We Are Made Of
How We Study Molecular Structure
Separation

How did chemists determine the thousands of structural formulas that we use today to describe naturally occurring molecules? This question is far too complex to be discussed here in detail. However, since the separation of compounds, mixture analysis, and molecular Structure determination remain central to modern biochemistry, we provide a "mini-review" below, along with A number of References to help the reader navigate the literature on these topics.

Before determining The structure of a given substance, it must first be isolated in pure form from the complex mixtures found within Cells and Tissues, where the concentration of the target substance is often very low. Next, the molecules must be degraded, and the resulting fragments separated, purified, and identified. Precise quantitative analysis is required to determine the relative amounts of the components. Finally, considerable ingenuity is needed to reassemble all the fragments and establish the STRUCTURE OF THE original molecules.

a. Cell Fractionation [105–110]

The Starting Material for fractionation can be fresh tissue or a pellet of packed microbial cells, typically obtained by centrifugation.

Tissue is disrupted either in a meat grinder or, for gentler Treatment, in a homogenizer¹. Microbial cells are most commonly broken down by sonication or high-pressure extrusion. During fractionation, it is crucial to select the appropriate pH and buffer composition, and—when isolating subcellular Organelles—the correct osmotic pressure. To maintain organelle integrity, 0.25 M sucrose is frequently used as a suspending medium, supplemented with MgCl₂ and a metal-chelating agent such as ethylenediaminetetraacetic acid (EDTA) (Table 4-2). Soluble Enzymes are generally extracted without The addition of sucrose, but using reducing agents such as Glutathione (Box 7-B), mercaptoethanol, or dithiothreitol (Section 3.3.a).

The resulting crude homogenate is strained and usually centrifuged briefly to remove cell debris and other particulate "fragments." Cell organelles are typically isolated by differential centrifugation [106, 107]. One standard protocol is as follows. The homogenate in 0.25 M sucrose (which is isotonic to most cells) is centrifuged for 10 min at 600–1000 g to pellet nuclei and intact cells. The supernatant is then centrifuged for another 10 min at ~10,000 g to sediment Mitochondria and Lysosomes. Finally, centrifugation for one hour at ~100,000 g yields the microsomal pellet. Each separated fraction can then be resuspended and re-centrifuged to obtain a purer preparation of a specific organelle type. In many cases, pelleted particles are solubilized using chemical treatments, such as the addition of detergents. The supernatant remaining after high-speed centrifugation serves as the starting material for isolating soluble enzymes and numerous low-molecular-weight compounds.

¹ The most widely used homogenizer consists of a cylindrical vessel fitted with a tightly rotating Glass or plastic pestle.

b. Separation Based on Differences in Solubility

Certain Fibrous Proteins are virtually insoluble in Water, allowing all other sample components to be removed simply by dissolution. Soluble proteins are most frequently precipitated from aqueous solutions by salting out, which involves adding a high concentration of a salt such as ammonium sulfate. Different proteins precipitate at different salt concentrations; thus, a protein fraction precipitating within a specific concentration range can be isolated and subsequently purified further. Methods based on such fractional precipitation are widely used as an initial purification step because they allow large quantities of material to be processed simultaneously.

RNA is typically obtained by treating the preparation with water-saturated phenol, which precipitates all proteins. Under certain conditions, DNA can also be removed. Successive stages of precipitation and extraction allow RNA to be separated from Polysaccharides (remaining in the aqueous layer), DNA (if present), and other components [111–113]. Nucleic Acids can be separated from proteins by digesting the latter with Proteolytic Enzymes.

Certain CARBOHYDRATES, such as Cellulose and Glycogen, are resistant to boiling alkali, which makes it possible to eliminate all Other components of the mixture. Lipids are extracted from tissues using nonpolar Solvents [114], such as a 2:1 mixture of CHCl₃ and CH₃OH.

c. Separation Based on Partitioning Between Different Phases

Many of the most important Separation Methods rely on the repeated partitioning of compounds between two different phases, at least one of which is typically liquid. Small molecules can be separated by countercurrent distribution, where a sample is repeatedly equilibrated between two liquid phases of differing polarity. Following each equilibration, fresh portions of both liquids are introduced in a "countercurrent" manner using specialized apparatus [115].

Alternatively, a powder or finely divided solid can serve as one of the phases, packed into a vertical Column or coated in a thin layer on a glass plate. All such methods are termed chromatographic—a term coined by M. Tsvet, who in 1903 first described the separation of plant leaf pigments (chlorophylls and carotenes). By passing pigments dissolved in nonpolar solvents (such as hexane) through a column packed with aluminum oxide or other adsorbents, Tsvet discovered that the mixture separated into colored bands migrating down the column as the solvent flowed. By continuously passing solvent through the column, individual pigments could be eluted in pure form. This method, known as adsorption chromatography, remains widely used today. In this technique, pigments do not dissolve in the solid material but are instead adsorbed onto its surface.

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FIG. 2-34. Two-dimensional Thin-Layer Chromatography (on silica gel) of a flavin mixture obtained by the irradiation of vitamin riboflavin. Light spots on the image are due to the fluorescence of compounds under ultraviolet light. A portion of the riboflavin remains unchanged (spot designated RF). Arrows indicate the sample origin. Solvent systems used were acetic acid/2-butanone/methanol/benzene for the first dimension, and n-butanol/acetic acid/water for the second [147].

Silica gel containing high water content is frequently used as a Column packing material; in this case, component separation is driven by partitioning between the aqueous phase immobilized on the silica gel and the water flowing through the column. Reversed-phase columns are used for lipid separation, packed with silica gel, aluminum oxide, or another inert material impregnated with a nonpolar liquid. Here, the Mobile phase acts as a more polar solvent.

Paper chromatography relies on high-quality filter paper that adsorbs water as the stationary phase. In recent years, thin-layer chromatography (TLC) has become widespread; it replaces paper with a thin layer of silica gel coated on a glass plate. This method is much more convenient than paper chromatography, yielding faster and higher-resolution separations (Fig. 2-34). Volatile substances are separated by gas chromatography, which is based on establishing a dynamic equilibrium between a gas phase and a stationary phase at relatively high temperatures. The applicability of this method is expanding due to the feasibility of converting analytes into volatile derivatives (such as sugar methyl esters or trimethylsilyl derivatives).

Affinity Chromatography plays a particularly vital role [116, 117]. It is based on specialized adsorbents that interact specifically with macromolecules, selectively retaining a particular type of macromolecule (hence the name of the method). An example, previously mentioned in Section G.10, is the adsorption of complementary nucleic acid fragments onto immobilized DNA. Affinity chromatography is also employed to purify enzymes, Antibodies, and other proteins capable of binding tightly to specific small molecules.

The open porous structure of gels formed by agarose derivatives (Fig. 2-18) makes coarse-grained agarose powders a convenient solid support for preparing adsorbents. The hydroxyl groups of agarose are frequently modified by attaching amines. First, agarose is treated with Cyanogen bromide (Br—C ≡ N) in an alkaline medium, thereby "activating" the carbohydrate chain (the chemical details of this process are discussed in reference [118]), after which amines are added. The overall reaction is described by the following equation:

Adsorbents containing A wide variety of R-groups can be prepared in this manner. Furthermore, by attaching a diamine to the activated agarose [R = (CH₂)_n—NH₂], the resulting ω-aminoalkyl agarose can be coupled to Other Compounds. This is accomplished using a water-soluble carbodiimide reaction;

Carbodiimides are widely used in laboratory practice to form amide and phosphodiester bonds. The amide bond formation depicted in equation (2-18) can be illustrated by scheme (2-19). For reactions carried out in nonaqueous media, dicyclohexylcarbodiimide (R" = cyclohexyl; see scheme) is frequently used; however, for subsequent attachment of groups to polysaccharides, it is preferable to employ a water-soluble reagent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [119].

In addition to those described above, many other methods are known for preparing adsorbents for affinity chromatography.

An example of using affinity chromatography for Enzyme Purification is the isolation of staphylococcal nuclease (an enzyme that hydrolyzes DNA) via column chromatography on agarose containing the following group [120]:

d. Separation Based on Molecular Size

Dialysis [121] and ultrafiltration [122] rely on a thin semipermeable membrane (such as cellulose acetate or cellophane) with pores 1–10 nm in diameter (most commonly 5 nm) acting as a barrier. Such membranes allow small molecules to pass through while retaining large ones. Dialysis is driven by diffusion and can be accelerated by stirring the solution, whereas the filtration rate depends on the pressure differential across the membrane. Gel filtration [123] operates on a more complex principle. A column is packed with a dextran-like material with a high degree of cross-linking (Sephadex¹), typically in the form of compressed, soft beads. Each bead consists of a three-dimensional network of carbohydrate chains (Fig. 2-18). The spaces between these chains—determined by the number of cross-links formed during the chemical Processing of the gel—are too small for large molecules to enter, yet perfectly sized to trap smaller molecules. When a mixture of various molecules is passed through such a column, the small molecules diffuse into the gel particles and are retarded, whereas the large molecules pass through unhindered (Fig. 2-35). Sephadex G-25 retains nothing except low-molecular-weight salts and simple cyclic sugars. Sephadex G-200, which has a much lower degree of cross-linking, is used to separate macromolecules with molecular weights ranging from 5,000 to 200,000.

¹ A commercial product of Pharmacia Fine Chemicals, Uppsala, Sweden.

FIG. 2-35. Separation of Oligosaccharides by gel filtration. Sugars dissolved in distilled water were passed through a column packed with Sephadex G-25. The peaks (from right to left) correspond to glucose, cellobiose, cellotriose, etc. (Flodin P., Aspberg K., Biol. Struct. Funct. 1st., Proc. IUB/IUBS Int. Symp., 1960, 1, 345, 1961.)

e. Centrifugation

A wide variety of separation methods involve The Use of preparative centrifuges [124, 125]. The Sedimentation Rate of molecules in a centrifugal field depends on their shape and size. Each molecule is characterized by its sedimentation coefficient, s, expressed in Svedberg units, S. However, once Sedimentation Equilibrium is established in the centrifuge, particle density becomes the primary factor determining their final position.

For rough preliminary separation of cellular fragments, brief sequential centrifugation at several different speeds (with varying centrifugal acceleration) is sufficient (Section 3.1.a). However, to obtain the highest purity preparations of organelles or molecules, density gradient centrifugation is employed. For example, to separate RNA into several fractions differing in sedimentation coefficients, a sucrose concentration gradient (ranging from 25% at the bottom to 5% at the top) is first created in a plastic centrifuge tube. The RNA preparation is then carefully layered on top, and centrifugation is carried out at very high speed for several hours. The RNA preparation separates into a series of sharply defined, slowly sedimenting bands stabilized by the sucrose gradient. The tube is subsequently punctured from the bottom, and fractions are collected drop by drop into test tubes using a fraction collector. THE POSITION OF each fraction and its RNA content are then determined.

With a sufficiently long centrifugation time, particles reach equilibrium positions within the density gradient. It is precisely in this manner, according to their density in a sucrose gradient, that cellular organelles are separated (Chapter 1, Section B.6). Macromolecules (such as DNA) are most commonly separated in a CsCl gradient [126]. Density gradients are quite stable in concentrated salt solutions, and very strong centrifugal fields are applied to achieve optimal separation. Consequently, single-stranded DNA can be separated from double-stranded DNA, or DNA preparations with varying GC contents can be resolved. The latter is based on differences in the buoyant density ρ of such DNAs in a CsCl solution, which varies with GC content According to the following approximate equation:

ρ = 1.660 + 0.098 ∙ (mole fraction of GC pairs).

f. Electrical Charge

Several separation methods are based on differences in the net electrical charge that molecules carry at a given medium pH. For many compounds, this net charge can be easily estimated from the number of acidic and basic groups they contain. Let us assume that the pKa values of each group and its degree of dissociation at a given pH are known. At a specific pH, known as the isoelectric point, the net charge of the molecule becomes zero, and it does not move in an electric field. At any other pH value, the molecule will migrate toward the anode (+) or cathode (–).

Electrophoresis—The process of separating molecules based on their differing migration rates in an electric field—is carried out using a wide variety of techniques. A very small volume of a solution containing a mixture of proteins (such as Blood Serum proteins) is applied as a thin strip to a sheet of filter paper or cellulose acetate. The sheet is saturated with a buffer, and an electric current is passed through it. A voltage of several hundred volts is sufficient to separate serum proteins within 1 hour. To accelerate the process and minimize the diffusion of low-molecular-weight substances, high-voltage electrophoresis is widely used, with applied voltages ranging from 2 to 3 kilovolts. The sample is continuously cooled using thermostated plates; sometimes the entire system is immersed in a vessel of kerosene for the same purpose. Preparative electrophoretic separations of large quantities of material are performed in flat trays filled with starch or other gels. One of the most common and sensitive methods for protein separation is Polyacrylamide gel electrophoresis in a column. This method, now highly refined, allows molecules to be separated simultaneously by size and electrical charge; it is known as the electrophoretic molecular sieve technique [127, 128].

In isoelectric focusing within a vertical column, a pH gradient is electrochemically established between the anode and cathode. This pH gradient is stabilized by a density gradient, most commonly sucrose, and the entire system is carefully thermostated. Proteins within the column migrate toward their respective isoelectric points, where they "focus" into extremely narrow bands. Two adjacent bands may be separated by an interval of only 0.01 pH units. It is generally accepted that under normal experimental conditions, the isoelectric points of proteins are close to their isoionic points—that is, the pH values at which proteins remain isoelectric in the complete absence of added electrolytes [129].

An exceptionally important separation technique dependent on the presence of charged electrical groups is Ion-exchange chromatography. Typically, aqueous solutions of mixtures are used along with columns packed with an ion-exchange resin—a porous material containing bound ionic groups, such as –SO₃^–, –COO^–, –NH₃⁺, or quaternary ammonium groups. For the separation of small molecules, synthetic resins based on cross-linked polystyrene are generally employed. For large molecules, cellulose derivatives or cross-linked dextrans (Sephadex) are more suitable. Compounds with positively charged groups, such as Amino Acids in an acidic solution, are applied to a cation-exchange resin column (e.g., Dowex 50), which contains dissociated sulfonic acid groups. The adsorbed Amino acids are then eluted with an HCl solution or a buffer of increasing pH. This type of process forms the basis for the automated quantitative analysis of amino acid mixtures derived from the Hydrolysis of Proteins or Peptides (Fig. 2-36). Purine and pyrimidine bases can also be separated on sulfonate-substituted polystyrene. Negatively charged NUCLEOTIDES are typically separated on resins containing quaternary ammonium bases [130]. A modified version of this method has been developed for separating very small amounts of material; for example, the content of each nucleotide can be quantitatively determined in a rat Liver mitochondrial preparation containing 5–8 mg of protein [131].

FIG. 2-36. Results of ion-exchange chromatography of an amino acid mixture using an amino acid analyzer. An amino acid mixture (0.5 nmol of each) was applied to a column 0.9 cm in diameter and 58 cm long, packed with fine polystyrene sulfonate particles (Durrum type DC-4A), and eluted with citrate buffer at three successively increasing pH values from 3.5 to 6.4. The effluent passing through the column was treated with fluorescamine (p. 180), and the fluorescence of the resulting product was continuously recorded. (Courtesy of Durrum Chemical Corp.)