Biotinylation

Overview

In biochemistry, biotinylation is the process of covalently attaching a biotin tag to a molecule or surface.

Labeling methods

Primary amine biotinylation

The most common target for modifying protein molecules is amine groups, which are present as lysine side chain α-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 solutions. For reactions in aqueous solution, they must first be dissolved in an organic solvent, then diluted into the aqueous reaction mixture. The most commonly used organic solvents for this purpose are dimethyl sulfoxide (DMSO) and [dimethyl formamide]] (DMF), which are compatible with most proteins at low concentrations. NHS-esters of biotin are also membrane permeable, thus they may be used for biotinylating internal as well as external components of a cell.

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 is from the sulfonate group on the N-hydroxysuccinimide 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 plasma membrane.

Reaction chemistries of NHS- and sulfo-NHS-esters are essentially identical: an amide bond is formed and NHS or sulfo-NHS become leaving groups. Because the target for the ester is deprotonated primary amines, the reaction is significant above pH 7. Hydrolysis of the NHS-ester is a major competing reaction, and the rate of hydrolysis increases with increasing pH. NHS- and sulfo-NHS-esters have a half-life 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.

Purpose

Purification

The biotin tag can be used in affinity chromatography together with a column that has avidin (also streptavidin or Neutravidin) bound to it, which is the natural chelator for biotin.

Detection

This tag can also be used in detection of the protein via anti-biotin antibodies or avidin/streptavidin tagged detectors like horseradish peroxidase or a fluorescent dye. This can be useful in localization, ELISA assays, ELISPOT assays, western blots 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 bond. This very tight binding makes labeling proteins with biotin a useful tool for applications such as affinity chromatography 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 that 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.