Crosslinking reagents or crosslinkers are used to covalently bind two or more protein molecules to facilitate the identification of relationships between near-neighbor proteins, ligand-receptor interactions, three-dimensional protein structures, and molecular associations in cell membranes. In the same manner, they can also be used to modify nucleic acids, drugs, and solid surfaces, and in the preparation of antibody-enzyme conjugates and immunotoxins.
How can these reagents do all these? Simple. Protein crosslinking reagents typically contain two or more chemically reactive groups that will connect themselves to the functional groups (e.g. primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids) found in proteins and other molecules. These reactions make the molecules stable enough to allow for intensive scientific analysis.
Types of Crosslinking Reagents
There are three different types of crosslinkers – homobifunctional, heterobifunctional, and photoreactive crosslinking reagents. How do these types of crosslinkers differ from one another and how do you know which one to use for your specific application? Here are some things that you may need to consider when choosing a suitable crosslinker.
Homobifunctional crosslinking reagents have identical reactive groups at either ends and are generally used in binding like functional groups. These reagents are mainly used to form intramolecular crosslinks and can be used in the preparation of polymers from monomers. While this type of reagents can capture a general snapshot of all protein interactions, they cannot provide the accuracy required for other types of crosslinking applications.
Some common examples of amine-to-amine crosslinkers include disuccinimidyl suberate or DSS (ideal for receptor ligand cross-linking), disuccinimidyl tartrate or DST (used for applications wherein crosslink cleavability is required while keeping the protein disulfide bonds intact) and dithiobis succinimidyl propionate, or DSP (ideally used for crosslinking intracellular proteins prior to cell lysis and immunoprecipitation as well as for fixing protein interactions prior to identification of weak or transient protein interactions). Some common examples of sulfhydryl-to-sulfhydryl crosslinkers include BMOE and DTME.
Heterobifunctional crosslinking reagents possess two different reactive groups and can be used to link dissimilar functional groups. These reagents are used to produce multiple intermolecular crosslinks and conjugates using dissimilar biomolecules. Unlike homobifunctional crosslinkers that merely facilitates single-step conjugation of molecules, heterobifunctional crosslinkers allow for two-stage conjugations. This minimizes undesirable polymerization or self-conjugation.
Some common examples of heterobifunctional crosslinkers include MDS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), GMBS (N-γ-Maleimidobutyryloxysuccinimide ester), EMCS (N-(ε-Maleimidocaproyloxy) succinimide ester) and sulfo-EMCS (N-(ε-Maleimidocaproyloxy) sulfo succinimide ester), to name a few.
Photoreactive crosslinking reagents are heterobifunctional crosslinkers that become reactive only upon exposure to ultraviolet or visible light. This type of crosslinking reagent is best used for non-specific bioconjugation and can be used to bind nucleic acids, proteins and other molecular structures. There are two photoreactive chemical groups that are being widely used in protein laboratories worldwide – aryl-azides and diazirines.
Aryl azides (N-((2-pyridyldithio)ethyl)-4-azidosalicylamide) are the most widely used photoreactive reagents in crosslinking reactions. Upon exposure to 250-350nm UV light, these reagents can facilitate the formation of a nitrene group that may set off an addition reaction with the double bonds. Additionally, these crosslinkers may initiate the production of C-H insertion products or react with a nucleophile. Some common crosslinking reagents that belong to this group include ANB-NOS (N-5-Azido-2-nitrobenzyloxysuccinimide) and Sulfo-SANPAH.
On the other hand, NHS-ester diazirines or azipentanoates contain a photoactivatable diazirine ring and an N-hydroxysuccinimide (NHS) ester which efficiently reacts with primary amino groups in neutral to basic buffers (pH 7 to 9) to form stable amide bonds. It exhibits better photostability as compared to the phenyl azide group and can be easily activated with long-wave ultraviolet light (330 to 370nm) to produce carbene intermediates that form covalent bonds with any peptide backbones or amino acid side chains within the spacer arm distance.
