Part IV: Chemical Disruption Methods
It is very possible to disrupt a biological sample using nothing more than water and a blender. This would be considered a mechanical approach to sample disruption. However, most methods use lysis buffers/solutions instead of water as they provide a degree of stability when isolating specific biomolecules. Virtually all lysis solutions address pH (which is why they are usually called lysis buffers) but they may also control ionic strength, osmotic strength, and the activity of nucleases and proteases. When isolating membrane proteins, surfactants are normally used to partition the membrane proteins from the membrane to surfactant particles, called micelles. Other common lysis buffer additives include lytic enzymes, which can liberate cellular contents from cell wall envelopes, and chaotropes that disrupt the ordered structure of biological systems which protects biomolecules from enzymatic degradation.
Lysis buffers in many instances can be used to lyse cells and tissues without the assistance of mechanical homogenizers. Indeed one of the most common disruption methods relies on lysing Escherichia coli with an alkaline solution of SDS (the detergent sodium dodecylsulfate) for plasmid isolation. Similarly, adherent tissue culture cells can be lysed with high concentrations of chaotropic guanidine salts (e.g., chloride or isothiocyanate). For solid and resilient samples, lysis buffers are commonly used in combination with a mechanical disruption method. This is particularly true for tissues which are very dense, like organs and seeds. Some microorganisms which are resistant to chemical and enzymatic lysis, such as members of the genus Mycobacterium, must also be disrupted mechanically.
Other additives protect liberated biomolecules from denaturation, oxidation and enzymatic degradation. Reducing agents protect free thiol groups from oxidation, especially cysteine located in the active site. Protease inhibitors are regularly added to prevent protein degradation from proteases released from cells during homogenization.
Surfactants, which are commonly called detergents, have the characteristic of disrupting the distinct interface between hydrophobic and hydrophilic systems. Biological membranes, the most obvious hydrophobic/hydrophilic interfaces, are the primary target of detergents. Indeed with the example of E. coli and SDS, the detergent completely (and effectively) obliterates the distinct interface separating the cell from its environment, i.e., the membrane. However, SDS also has the ability to unfold (denature) cytosolic proteins and partition membrane proteins into small detergent droplets (micelles). Depending upon the detergent used and its concentration, the impact these surfactants have on biological systems will vary greatly.
Detergents have at least two fundamental properties, namely a water soluble hydrophilic head and a hydrophobic (oil soluble) tail. These properties allow detergents to insert into and then disperse membranes, in addition to unfolding proteins. Depending upon the chemical makeup of the hydrophilic and hydrophobic ends its action on proteins and membranes will vary. Not all surfactants are chemically equal as some are capable of completely solubilizing membranes and denaturing proteins while others, like mild surfactants, will disassociate loosely bound proteins.
A major characteristic of surfactants is whether the hydrophilic group is ionic or non-ionic. Ionic surfactants tend to be better at solubilizing membranes and denaturing proteins. With ionic surfactants, the hydrophilic moiety is typically a sulfate or carboxylic group for anionic surfactants or ammonium group for cationic surfactants. SDS (sodium dodecyl sulfate), is an anionic detergent with a sulfate hydrophilic head and 12 carbon tail (dodecyl or lauryl) which is important not only in the lab but also in many household detergents. Sodium deoxycholate is a carboxylic based anionic detergent derived from bile salts which is commonly used in many lysis buffers. CTAB (cetyltrimethylammonium bromide) is a cationic detergent widely used in the isolation of DNA from plants.
In addition to detergents with a net charge, zwitterionic detergents are a class of surfactants that possess both anionic and cationic groups and have a net charge of zero. The zwitterionic detergent CHAPS, a derivative of cholic acid, is effectively used for isolating membrane proteins.
Non-ionizing detergents have a head which is polar, but uncharged, such as a glycoside (sugar) or polyethylene chain, tend to be milder and less likely to denature proteins, but still capable of dispersing some membranes. They often act to dissociate loosely interacting molecules. These surfactants, such as Triton X-100, Brij-35, NP-40 and Nonidet P-40, are widely used in immunoassay wash solutions at low concentrations, but also in lysis buffers at higher concentrations.
The value of detergents when applied to isolating membrane proteins is related to their ability to form micelles. In aqueous environments and at the correct concentrations, detergents will spontaneously form small particles called micelles where the hydrophilic heads orient outward and the hydrophobic tails congregate inwards. The concentration at which this occurs is called “critical micelle concentration” or CMC. Depending upon the detergent, the molecular weight of the micelles can range from 1,200 to 80,000 daltons.
Micelles dissolve into and disrupt cell membranes which then disperse into membrane/micelle hybrids particles. Proteins embedded in cellular membranes are picked up by these micelles. A constant equilibrium between monomer detergent molecules and micelles ideally lead to a dispersion of membrane proteins so that each micelle contains one protein. For protein purification, the protein/micelle acquires characteristics which allow it to be separated from other membrane proteins/micelles.
Detergents and their use are application specific and not always predictable.
Table 2. Detergents used for sample preparation and their properties.
|SDS (sodium dodecylsulfate)||Anionic||Strong detergent used to disrupt membranes and denature proteins||Commonly used between 1-10%|
|Sodium Deoxycholate||Anionic||Derived from bile salts. Effective at solubilizing proteins and disrupting protein-protein interactions.||Common use level is 0.5%.|
|CTAB (cetyltrimethylammonium bromide)||Cationic||Popular cationic detergent used for the isolation of DNA from plants. Polysaccharides associated with plants are insoluble in CTAB and high concentrations of NaCl. This can be used to effectively separate DNA from plant carbohydrates.||For DNA isolation buffers, typical use level is 2%.|
|NP-40 (nonyl phenoxypolyethoxyl ethanol)||Non-ionic||Generally mild surfactant which can dissolve cytoplasmic membranes but not nuclear membranes. Useful for isolating nuclei.||Use at 0.1 to 1%.|
|Nonidet P-40 (octylphenoxy polyethoxyethanol)²||Non-ionic||This mild surfactant is useful for disrupting cytoplasmic membranes of cultures cells, but lacks the strength to emulsify nuclear membranes. Consequently it can be used to harvest cytoplasmic proteins and analytes.||Use at 0.1 to 1%.(P)|
|Triton X-100||Non-ionic||This is a mild surfactant/surfactant that has polyethylene oxide as a hydrophilic group and a tetramethylbutyl phenyl group as the hydrophobic portion.||For lysis solutions, up to 5%. In wash solutions, 0.1-0.5%.|
|Polysorbate 20 (Tween 20, Polyoxyethylene (20) sorbitan monolaurate)||Non-ionic||This surfactant is a heavily modified sorbitol in which polyoxyethylenes serve as the hydrophilic group and a 12 carbon lauric acid as the hydrophobic end. It is a very biomolecule friendly surfactant, being used in foods, pharmaceuticals, and in wash solutions for assays.||Typically used at very low concentrations of 0.1%.|
Where surfactants are used to disrupt the interface between hydrophobic and hydrophilic systems, chaotropes are used to disrupt the weak interactions between molecules, like hydrogen bonding in water and hydrophobic interactions between proteins. Chaotropes are effective at denaturing proteins that can cause havoc on freshly homogenized samples, which is the rationale for adding chaotropes to RNA lysis buffers. Common chaotropes used in lysis buffers include sodium iodide, guanidine HCl, guanidine isothiocyanate, and urea.
Unlike surfactants which are used at relatively low concentrations, chaotropes are used at high molarities. Guanidine salts, used extensively for RNA isolation, is used at 6 M concentrations. Sodium iodide, which at times is used like guanidine, is also used at 6 M. Urea is often used at 9.5 M. Very often chaotropes are used in combination with detergents so that biological systems can not only be denatured, but emulsified as well.
Chaotropes are widely used and applicable to nucleic acid isolation procedures which use silica based resins/gels for purification. Nucleic acids liberated from tissues lysed in chaotropic agents, such as 6M guanidine, supplemented with Proteinase K (an unusually hearty protease that is active in both denaturing conditions and elevated temperatures) will adsorb to silica gel upon the addition of ethanol. The very clean nucleic acids can be eluted with water or TE buffer.
Enzymatic treatment of tissues and cells can be a very effective first step in processes where cell walls and extracellular matrices may provide unwanted contaminants in a cell/tissue extract. On-the-one-hand, primary hepatocytes can be generated from sacrificed rats where the liver has been perfused with a combination of trypsin and collagenase. This allows for the harvesting of viable, intact cells from a tissue that releases vast amounts of proteolytic enzymes when homogenized mechanically. Similarly, yeast cells can be treated with cell wall degrading enzymes to yield protoplasts (naked cells) and cell wall shells, or what is commonly referred to as ghosts. This is a common step in traditional transformation procedures used with yeast, but it can also be used to selectively harvest periplasmic enzymes, cell wall mannans or similar component. Likewise, plants can be treated with cellulases to yield protoplast while filamentous fungi can be treated with chitinase.
Proteases are used in sample disruption to disaggregate tissues and release individual cells, or in the case of genomic DNA isolation, attack other proteins that may either bind up the DNA (histones) or threaten the final product (nucleases). Proteases such as trypsin, dispase, and collagenase are used to release cells from tissues and culture plates. However outside of this application, they are undesirable in disrupting samples as they also degrade receptors and other surface proteins. Proteinase K is extensively used in DNA isolation as it is resistant to SDS and heat, both which are used in the typical genomic DNA isolation procedure.
Highly specific lytic enzymes are very useful in sample preparation protocols. With plant, yeast, and molds, cell wall degrading enzymes can be used to either rupture cells in hypertonic buffers or generate protoplasts in isotonic lysis buffers. Most cell wall degrading enzyme preparations are a combination of enzymes as cells walls are typically composed of a mixture of polymers. Yeast cell walls contain glucans and mannans, molds contain chitin, glucan, and galactomannans, and plants have a combination of cellulose and xylans.
Lysis solutions using enzymes at a minimum require a buffer. When generating protoplasts, cells are typically treated with buffered enzyme in the presence of an osmotic stabilizer, such as 1 M sorbitol. The enzymes tend to degrade holes in the cell wall which then allow the protoplast to escape. Gentle centrifugation of protoplasts allows for the separation of empty shells from the cell membrane and its contents. This can be an effective method of separating periplasmic enzymes from other cell associated proteins.
Aside from buffers, there are several other important components of homogenization buffers that warrant discussing. When the objective is to purify an active protein, homogenization buffers may contain many additional components. These can generally be viewed as additives that will help to retain the active form of the protein and those that prevent the degradation of the protein.
The cytosol contains high concentrations of solutes in a reduced environment. Protein concentrations have been estimated to be as high as 30 mg/ml. Liberating the contents of cytosols causes the solutes to become rapidly diluted which not only can cause non-associated solutes to diffuse, but also protein subunits and co-factors. Osmotic stabilizers, such as sucrose or sorbitol, can be added to help bind up water and prevent dissociation of related solutes.
With many homogenization methods, high volumes of air are also introduced into the system, where the oxygen can shift the environment from reduced to oxidized. Within cells, oxygen tension is extremely low, essentially anaerobic, thus the introduction of oxygen and their related radicals can lead to deleterious effects. Reducing agents such as glutathione, dithiothreitol, and ß-mercaptoethanol can react with oxidized species and prevent their negative consequences.
For protein isolation procedures, it is very common to add protease inhibitors to the homogenization buffer and/or homogenate. In animal cells, proteases are contained in lysosomes where their function is to recycle the amino acids and breakdown foreign material. Dependent upon the cell and tissue type, the concentration lysosomes and associated enzymes can be high. The proteases contained within are heterogeneous and capable of degrading proteins at many different locations. Generally there are exoproteases which cleave both the amino and carboxyl terminal residues, as well as endoproteases which can attack specific peptide bonds.
Plants are generally believed to lack lysosomes, but rather use vacuoles in much the same manner. Microorganisms also have proteases, but these are usually located in the periplasmic space. Similar to animal cells, disruption of plant and microbial cells releases the proteases which then can degrade proteins in the homogenate.
The deleterious action of proteases can be reduced by keeping samples cold while processing and by adding protease inhibitors. Though it is impractical to inhibit every type of proteases, several inhibitors can drastically reduce proteolytic activity. These inhibitors are summarized in Table 3.
Table 3. Commonly used protease inhibitors used during sample processing.
|PMSF||Cysteine and serine proteases||0.1 – 1 mM (24-240 µg/ml)||PMSF has a short half life in water. Dissolve in ethanol and add just before processing.|
|EDTA||Metalloproteases||1 mM (0.37 mg/ml)||EDTA chelates divalent cations which are required by metalloproteases.|
|Pepstatin A||Acid Proteases (aspartyl peptidase)||1-2 µM (0.5-1.0 µg/ml||Prepare 1 mg/ml in ethanol (stable at -20°C). Use by diluting 1 µl/ml.|
|Leupeptin||Serine and thiol proteases||10-100 µM (5-50 µg/ml)||Prepare 25 mg/ml stock and use 0.2-2 µl/ml.|
|Aprotinin||Serine proteases||0.1-0.8 µM (0.65-5.2 µg/ml)||Use 1 µl/ml of 5 mg/ml stock in water (0.76 mM).|
When homogenizing plants, the disruption of the vacuole is much like disrupting a lysosome. Proteases are released in addition to phenolic oxidases. Plants contain substantial concentrations of phenolic compounds which when oxidized can react with proteins. Plant homogenate turns black as a result of these reactions. The addition of a phenolic scavenging reagent, such as polyvinylpyrrolidone used at a concentration of 0.5-2%, can bind the phenolics and prevent their oxidation and subsequent reaction with proteins.
¹ The terms surfactant and detergent are used very loosely throughout scientific literature (including here). As surfactants are defined as chemicals that interact with hydrophobic/hydrophilic interfaces, detergent is chemically defined as an alkylbenzylsulfonate, a specific subgroup of surfactant.
² Nonidet P-40 is often designated NP-40 which is a chemically similar but different surfactant.
(P) indicates that there is an associated Protocol.