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Webpage Updated: February, 2024
Updated Webpage: This is the revised version of "Part IV: Chemical Disruption Methods of Guide to the Disruption of Biological Samples - 2012)", the original was archived February 2024 (link).
Chemicals, such as surfactants, detergents, and chaotropes, can be a gentler, less energy-intensive way of cell lysis than purely mechanical or physical methods. Such chemicals can thoroughly disrupt cells, make the cell membrane permeable or under appropriate conditions enter the cell to selectively release molecules. This allows for membrane proteins to be extracted intact, which can otherwise be denatured and destroyed with mechanical lysis.
Detergents are effective at dissolving cell membranes and liberating the cytoplasm. Apart from washing away the cell membranes, their usage also creates a dynamic system which can stabilize proteins (Neugebauer, 1990). When isolating membrane proteins, detergents are used to partition the membrane proteins from the lipid layer to emulsified particles, called micelles. This is considered a gentle lysis. In contrast, harsher detergents can fully rupture cells and denature proteins. Such lysis procedures, e.g. with an alkaline solution of SDS (the detergent sodium dodecyl sulfate), are used in the isolation of plasmids from bacteria.
Chaotrope agents disrupt the lattice of hydrogen bonding in water and therefore efficiently disorder biological systems which rely on water for stability (Timson, 2020). When used on cells, chaotropes disrupt the polar heads of the lipid bilayer and cause integral membrane proteins to unfold. Hence, chaotropes are considered a harsh but particularly efficient lysis method to inactivate enzymes such as Rnase, which can readily degrade analytes following cell lysis. Cells that lack walls, such as adherent tissue culture cells, can easily be lysed with high concentrations of chaotropic salts (e.g., guanidine isothiocyanate).
Surfactants (surface active compounds) are defined as chemicals that reduce the surface tension between solutions or phases. This class of chemicals includes detergents, which are the primary type of surfactant used in sample processing (Arachnea et al., 2012).
Detergents are amphipathic molecules, i.e. they have a water-soluble hydrophilic head and a hydrophobic (oil-soluble) tail. This property enabled detergents to insert into and then disperse membranes, thereby releasing membrane bound proteins. The specific action of the detergent on a protein and membrane varies depending upon the chemical makeup of the hydrophilic and hydrophobic ends. Some surfactants are capable of completely solubilizing membranes and denaturing proteins while others, more 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 an ammonium group for cationic surfactants. One of the most used surfactants for lysis is sodium dodecyl sulfate (SDS), an anionic detergent with a sulfate hydrophilic head and 12-carbon tail (dodecyl or lauryl). SDS is a common ingredient in household chemicals like cleaning agents, paints, or adhesives. Some other surfactants commonly utilized for cell lysis are sodium deoxycholate, a carboxylic-based anionic detergent derived from bile, and cetyltrimethylammonium bromide (CTAB), a cationic detergent which is widely used in the isolation of DNA from plants.
By design, 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. Detergents can have a mild or harsh effect on this interface. With weak detergents, such as Triton X-100, the surface tension is reduced between water and biomolecules, but not to the extent that proteins become unfolded, or denatured. In contrast, harsher detergents like SDS, can fully unfold proteins and are therefore more suitable for the isolation of other cell compounds like nucleic acids. An example therefore is the isolation of plasmids from E. coli where an efficient and complete rupture of the cell membrane is necessary. Importantly, the impact of any surfactant on a given biological system will vary with concentrations and conditions applied.
The ability of detergents to isolate membrane proteins is related to their micelle forming capabilities (Figure 1). In aqueous environments and at suitable 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 such micelles can range from 1,200 to 80,000 Daltons.
Figure 1. Chemical structure of a strong ionic detergent, such as sodium dodecyl sulfate, showing the hydrophobic and hydrophilic sections and micelle formation. SDS exists as individual monomers below critical micelle concentrations (CMC) while assembling as micelles above the CMC (Adapted from Life Canvas Technologies).
Micelles dissolve into and thereby disrupt cell membranes which then disperse into membrane-micelle hybrids particles. Proteins bound in cellular membranes are thereby integrated in these micelles. A constant equilibrium between monomer detergent molecules and micelles under ideal conditions leads to a dispersion of membrane proteins so that each micelle contains one protein (see Figure 2). These protein micelle complexes have specific properties that depend on the embedded protein, which allows for targeted protein purification and separation from other protein micelles in the mixture.
Figure 2. Solubilization of integral membrane proteins in micelles.
Ionic detergents with opposingly charged head groups, i.e., zwitterionic detergents, are classes 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 effective for isolating membrane proteins as it can extract them from the lipid bilayer and keep them in solution without denaturing and disrupting their native structure and function (Hjelmeland, 1980). CHAPS micelles have a small molecular weight (6150 g/mol) and a high critical micelle concentration (6-10 mM). Due to their small size and high micellar concentration, individual proteins are associated with a single CHAPS micelle particle. This allows the properties of that protein micelle particle to be exploited for purification. For instance, CHAPS micelles lacking protein can be easily removed from the lysate by dialysis or gel filtration. This allows for the purification of membrane proteins and subsequent removal of potentially interfering detergent molecules.
Non-ionizing detergents are uncharged but have a polar head and hydrophobic chain, which is commonly made of a glycoside (sugar) or polyethylene. No-ionizing detergents act by dissociating loosely interacting molecules. They tend to be more mild and less likely to denature proteins, while still capable of dispersing some membranes. Non-ionizing detergents, 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.
Table 1 provides a summary of many common detergents used in sample preparation. The choice of detergent should be tailored to specific applications, relying on the cell type and the characteristics of the desired product.
Detergent | Type | Characteristics | Use Level |
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%. |
While surfactants are disrupting the interface between hydrophobic and hydrophilic systems, chaotropes disrupt the weak interactions between and within molecules, like hydrogen bonding in water and hydrophobic interactions between proteins. Chaotropes are highly effective at denaturing proteins, and therefore commonly used in RNA lysis buffers as they immediately inhibit any present nucleases which would otherwise degrade RNA molecules before they can be extracted. Common chaotropes used in lysis buffers include sodium iodide, guanidine HCl, guanidine isothiocyanate, and urea.
Unlike surfactants which are used at low concentrations, chaotropes are used at high molarities. Guanidine salts or Sodium iodide, which are both extensively used for RNA isolation, are commonly applied at a molar concertation of 6 M. Urea is often used at 9.5 M. Chaotropes are frequently used in combination with detergents, for biological systems to be denatured as well as instantly emulsified. However, guanidine isothiocyanate causes starch to solidify, which increases the co-precipitation of both starch and polysaccharides with RNA due to structural similarities. RNA isolation techniques based on this extraction method may therefore not be able to effectively extract RNA from plant tissues with a high starch content (Wangsomnuk et al., 2016).
Chaotropes are widely applied to nucleic acid isolation procedures which use silica-based resins or gels for purification. Nucleic acids liberated from tissues lysed with chaotropic agents, such as 6M guanidine supplemented with the denaturation- and temperature-robust Proteinase K, will adsorb to silica gel upon the addition of ethanol. Purified nucleic acids can be eluted with water or TE buffer.
Chaotropes are widely used in protein purification schemes using ammonium sulfate, however the main advantage of chaotropes in sample disruption is their capacity to lyse cells. This capacity for cell lysis is based on the interference of chaotropes with the non-covalent interactions that keep the nuclear envelope and the cell membrane stable. Moreover, proteins that potentially interfere with the extraction of nucleic acids are denatured by chaotropes. However, for protein purification, chaotropes can denature proteins and are therefore most applicable to isolating biomolecules that lack activity.
Chaotrope | Characteristics | Concentration | Reference |
Sodium iodide | Used for extraction of DNA. | Use at a molar concentration of 6 M. | Suh et al., 2001 |
Guanidine HCl | A strong denaturant and a potent chaotrope that makes hydrophobic compounds more soluble and reduces the activity of enzymes. | Proteins tend to become randomly coiled and lose their ordered structure at high guanidinium chloride concentrations (6 M). | Ferreira et al., 2001 |
Guanidine isothiocyanate | Removes intact RNA from most tissues and cultured cells. | Use 4 M to lyse mammalian cells and 6-8 M to denature proteins, nucleic acids, and cell walls of bacteria and yeast cells. | Davis et al., 1986 |
Urea | Forms strong hydrogen bonds with the peptide backbone of proteins. | Use 6-10 M to lyse cells. | Timson, 2020 |
Depending on the objective and sample type, solvents and alkali solutions can also be used to disrupt cells and release analytes. It has been well-established that microbial cells can be dissolved with common organic solvents like butanol and hot toluene. The latter works by dissolving hydrophobic components in the inner membrane of phospholipids, which is particularly useful in the lysis of Gram-negative bacteria (Middelberg, 1995). Organic solvents such ethyl acetate, ether, benzene, methanol, phenyl ethyl alcohol, and dimethyl sulfoxide (DMSO) can permeate cell walls and therefore commonly used in quick extractions for metabolite assays.
Solvents can emulsify membranes but also partition or extract targeted analytes. Using selective solvents is common practice prior to HPLC analysis, where it has multiple advantages compared to alternative homogenization methods. These include accessibility, affordability, and capacity for recycling of used solvents (Lee et al., 2019). However, organic solvents are flammable, and there are certain restrictions on the use of water-miscible organic solvents, primarily related to fire safety (Lee et al, 2019).
Alternatively, alkali solutions are used to extract analytes from cells. Saponification of the cell wall's lipids, which is the hydrolysis of fatty acids from glycerol, is the mechanism of alkaline lysis of membranes using alkalis such as hydroxide and hypochlorite. Alkaline lysis is therefore a common technique for preparing DNA extracts. This very aggressive but highly effective approach only works for target products that are not degraded at high pH. Proteins can also be extracted using alkali lysis, which is then followed by a neutralization step.
Alkali lysis is applicable for process-scale disruption and can be achieved at low cost and high effectiveness. However, the harsh nature of extreme pH may be detrimental to the analyte. It is useful for analytes that do not easily denature or deteriorate at high pH levels, which is a limited number. Additional drawbacks of this method are the common product contamination, low selectivity, and toxicity.
Certain biological samples, such as cultured cells and gram-negative bacteria, can be lysed chemically using detergents and/or chaotropes. Detergents, which can be harsh and mild, form micelles that emulsify membranes and form small particles that can capture membrane bound proteins. Strong ionic detergents can also denature many biological molecules, while non-ionic detergents can be used to separate loosely associated molecules. Chaotropes, conversely, disrupt the aqueous environment that can both disrupt hydrophobic/hydrophilic interfaces and the intra-molecular non-covalent bonds of proteins. Chaotropes, especially those with guanidine, are widely used for nucleic acid isolation procedures. Often chemical lysis buffers will contain a combination of detergents and chaotropes.
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