In
biochemistryBiochemistry is the study of the chemical processes in living organisms. It deals with the structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules....
,
biotinylation is the process of covalently attaching a
biotinBiotin, also known as vitamin H or B7, is a water-soluble B-complex vitamin which is composed of an ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
tag to a molecule or surface.
The most common targets for modifying
proteinProteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues...
molecules are amine groups, which are present as lysine side chain epsilon-amines and N-terminal α-amines. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility.
NHS-esters of biotin have poor solubility in aqueous
solutionIn chemistry, a solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. Gases may dissolve in liquids, for example, carbon dioxide or oxygen in water. Liquids may dissolve in other liquids. Gases can...
s.
In
biochemistryBiochemistry is the study of the chemical processes in living organisms. It deals with the structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules....
,
biotinylation is the process of covalently attaching a
biotinBiotin, also known as vitamin H or B7, is a water-soluble B-complex vitamin which is composed of an ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
tag to a molecule or surface.
Primary amine biotinylation
The most common targets for modifying
proteinProteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues...
molecules are amine groups, which are present as lysine side chain epsilon-amines and N-terminal α-amines. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility.
NHS-esters of biotin have poor solubility in aqueous
solutionIn chemistry, a solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. Gases may dissolve in liquids, for example, carbon dioxide or oxygen in water. Liquids may dissolve in other liquids. Gases can...
s. For reactions in aqueous solution, they must first be dissolved in an
organicAn organic compound is any member of a large class of chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of compounds such as carbonates, simple oxides of carbon and cyanides, as well as the allotropes of carbon, are considered inorganic...
solventA solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution.The most common solvent in everyday life is water. Most other commonly-used solvents are organic chemicals. These are called organic solvents...
, then diluted into the aqueous
reactionA chemical reaction is a process that leads to the transformation of one set of chemical substances to another. They are studied by chemists under a field of science called chemistry. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, often coming about...
mixture. The most commonly used organic solvents for this purpose are
dimethyl sulfoxideDimethyl sulfoxide is the chemical compound with the formula
2SO. It was first synthesized in 1866 by the Russian scientist Alexander Zaytsev, who reported his findings in a German chemistry journal in 1867...
(DMSO) and dimethyl formamide (DMF), which are compatible with most proteins at low concentrations. NHS-esters of biotin can also diffuse through the
cell membraneThe cell membrane is the biological membrane separating the interior of a cell from the outside environment....
, meaning that they may be used for biotinylating internal as well as external components of a
cellThe cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos...
.
Sulfo-NHS-esters of biotin are more soluble in water, and should be dissolved in water just before use because they hydrolyze easily. The water solubility of sulfo-NHS-esters stems from their
sulfonateA sulfonate is a salt or ester of a sulfonic acid. It contains the functional group R-SO
2O-.- Sulfonate salts:Anions with the general formula RSO
2O
− are called sulfonates. They are the conjugate bases of sulfonic acids with formula RSO
2OH...
group on the
N-hydroxysuccinimideN-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
ring and eliminates the need to dissolve the reagent in an organic solvent. Sulfo-NHS-esters of biotin also can be used as cell surface biotinylation reagents because they do not penetrate the
cell membraneThe cell membrane is the biological membrane separating the interior of a cell from the outside environment....
.
Chemical reactions of NHS- and sulfo-NHS-esters are essentially identical: an amide bond is formed and NHS or sulfo-NHS becoming leaving groups. Because the targets for the ester are deprotonated primary amines, the reaction is prevalent above pH 7.
HydrolysisHydrolysis is a chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions, which may go on to participate in further reactions. It is the type of reaction that is used to break down certain polymers, especially those made by step-growth polymerization...
of the NHS-ester is a major competing reaction, and the rate of hydrolysis increases with increasing
pHpH is a measure of the acidity or basicity of a solution. It is defined as the cologarithm of the activity of dissolved hydrogen ions . Hydrogen ion activity coefficients cannot be measured experimentally, so they are based on theoretical calculations...
. NHS- and sulfo-NHS-esters have a
half-lifeHalf-life is the period of time, for a substance undergoing decay, to decrease by half. The name originally was used to describe a characteristic of unstable atoms , but may apply to any quantity which follows a set-rate decay....
of several hours at pH 7, but only a few minutes at pH 9.
There is some flexibility in the conditions for conjugating NHS-esters to primary amines. Incubation temperatures can range from 4-37°C, pH values in the reaction range from 7-9, and incubation times range from a few minutes to 12 hours. Buffers containing amines (such as Tris or glycine) must be avoided because they compete with the reaction.
Purification
The biotin tag can be used in
affinity chromatographyAffinity chromatography is a method of separating biochemical mixtures, based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand...
together with a column that has
avidinAvidin is a tetrameric protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
(also
streptavidinStreptavidin is a 52,800 dalton tetrameric protein purified from the bacterium Streptomyces avidinii. It finds wide use in molecular biology through its extraordinarily strong affinity for biotin ; the dissociation constant of the biotin-streptavidin complex is on the order of ~10-15...
or
NeutravidinNeutrAvidin protein is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity...
) bound to it, which is the natural ligand for biotin.
Detection
This tag can also be used in detection of the protein via anti-biotin
antibodiesAntibodies are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses...
or avidin/streptavidin tagged detectors like
horseradish peroxidaseThe enzyme horseradish peroxidase , found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
or a
fluorescent dyeA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
. This can be useful in localization,
ELISAEnzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality...
assays,
ELISPOTThe Enzyme-linked immunosorbent spot assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983....
assays,
western blotThe western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide or by the 3-D structure of the protein...
s and other immunoanalytical methods.
Other uses
The non-covalent bond formed between biotin and avidin or streptavidin has a binding affinity that is higher than most antigen and antibody bonds and approaches the strength of a
covalent bondA covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds...
. This very tight binding makes labeling proteins with biotin a useful tool for applications such as
affinity chromatographyAffinity chromatography is a method of separating biochemical mixtures, based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand...
using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals. Biotinylated protein such as biotinylated bovine serum albumin (BSA) is used in solid-phase assays as a coating on the well surface in multiwell assay plates. Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity.
Determining extent of biotinylation
Reaction conditions for biotinylation are chosen such that the target molecule (e.g. an antibody) is labeled with enough biotin substituents to purify or detect the molecule, but not so much that the biotin interferes with the function of the molecule. The HABA dye(2-(4-hydroxyazobenzene) benzoic acid) method is used to determine the extent of biotinylation. HABA dye is bound to avidin and yields a characteristic absorbance. When biotin, in the form of biotinylated protein or other molecule, is introduced, it displaces the dye, resulting in a change in absorbance at 500 nm. The absorbance change is directly proportional to the level of biotin in the sample.