Photo-activatable (or photo-chemical) crosslinking reactions require energy from light to initiate. Photoreactive groups are chemically inert compounds that become reactive when exposed to ultraviolet or visible light. Practically all varieties of photoreactive groups used in reagents for crosslinking applications require exposure to ultraviolet light (UV light) for molecular activation. The following image provides an example of the type of instrument used with photoactivatable crosslinkers and UV-crosslinking applications.
Representative ultraviolet lamp. Thermo Scientific Pierce UV Lamps are compact, multifunctional ultraviolet lamps with 252 nm, 302 nm and 365 nm dial-selectable wavelength settings especially for use with photoactivatable crosslinkers and UV-crosslinking methods.
Photochemical reactive groups have certain advantages over strictly thermochemical reagents for crosslinking and labeling applications with biological samples and experiments. Most importantly, they make it possible to add reagents at an early step in an experiment and then to initiate crosslinking (by exposure to UV light) at some later step that coordinates with the particular biological condition of interest. Additionally, many of these groups will conjugate to any one of several common functional groups in proteins that they encounter during the brief time when they are activated. This feature makes them particularly useful in capturing protein interactions (see discussion below).
Photoreactive groups that have been incorporated into crosslinking and labeling compound for use in bioconjugate techniques include aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. The most useful of these for protein biology research are aryl azides and diazirines.
Selected photo-reactive chemical groups used in protein crosslinking. Traditionally, varieties of aryl azides (also called phenyl azides, top two rows) were the most widely used. Psoralen (bottom right) reacts almost exclusively with RNA or DNA, being used to label nucleic acids or to crosslink and investigate the interaction of proteins with nucleic acids. Diazirines (lower left) are a newer class of compounds that are growing in popularity and availability for protein research. Squiggle bonds represent a labeling reagent or one end of a crosslinker having the reactive group.
This 45-page guide is of value to the novice as well as those who have previous experience with crosslinking reagents. It begins with a basic discussion on crosslinking and the reagents that are used. The guide also contains a discussion on various applications where crosslinking has been applied, including the powerful label-transfer technique for identifying or confirming protein interactions. Crosslinking chemistry is addressed in an easy-to-follow format designed to convey the important information you need without getting lost in details. Each Pierce crosslinking reagent is shown along with its structure, molecular weight, spacer arm length and chemical reactivity. The handbook concludes with a list of excellent references on crosslinker use and a glossary of common crosslinking terms.
When an aryl azide is exposed to UV light (250 to 350 nm), it forms a nitrene group that can initiate addition reactions with double bonds, insertion into C–H and N–H sites, or subsequent ring expansion to react with a nucleophile (e.g., primary amines). The latter reaction path dominates when primary amines are present in the sample.
Thiol-containing reducing agents (e.g., DTT or 2-mercaptoethanol) must be avoided in the sample solution during all steps before and during photo-activation, because they reduce the azide functional group to an amine, preventing photo-activation. Reactions can be performed in a variety of amine-free buffer conditions. If working with heterobifunctional photoreactive crosslinkers, use buffers compatible with both reactive chemistries involved. Experiments must be performed in subdued light and/or with reaction vessels covered in foil until photoreaction is intended. Typically, photo-activation is accomplished with a hand-held UV lamp positioned close to the reaction solution and shining directly on it (i.e., not through glass or polypropylene) for several minutes.
Three basic forms of aryl azides exist: simple phenyl azides, hydroxyphenyl azides, and nitrophenyl azides. Generally, short-wavelength UV light (e.g., 254 nm; 265 to 275 nm) is needed to efficiently activate simple phenyl azides, while long-UV light (e.g, 365 nm; 300 to 460 nm) is sufficient for nitrophenyl azides. Because short-wave UV light can be damaging to other molecules, nitrophenyl azides are usually preferable for crosslinking experiments.
Aryl azide reaction scheme for light-activated photochemical conjugation. Squiggle bonds represent a labeling reagent or one end of a crosslinker having the phenyl azide reactive group; R represents a protein or other molecule that contains nucleophilic or active hydrogen groups. Bold arrows indicate the dominant pathway. Halogenated aryl azides react directly (without ring-expansion) from the activated nitrene state.
Bioconjugate Techniques, 3rd Edition (2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide in the field of bioconjugation.
Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master biomolecular crosslinking, labeling, and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference tool for researchers looking to develop novel conjugation strategies for entirely new applications. It also contains an extensive introduction to the field of bioconjugation that covers all of the major applications of the technology used in diverse scientific disciplines as well as containing tips for designing the optimal bioconjugate for any purpose.
Although homobifunctional aryl azide crosslinkers were commercially available in the past, they have limited utility compared to alternatives and are no longer sold (search literature for "BASED, bis-[β-(azidosalicylamido) ethyl] disulfide)"). Nearly all applications for aryl azide reagents involve heterobifunctional chemistries in which an aryl azide group is paired opposite a different type of reactive group, such as an amine-reactive NHS ester. These compounds are used for a variety of bait-and-prey strategies to investigate protein-protein interactions or protein-nucleic acid interactions.
1. Capture protein interactions
Heterobifunctional NHS-ester/aryl-azide crosslinkers are used in experiments to discover or analyze the conditions in which a particular protein interaction occurs.
Suppose a researcher has a purified protein (X) and wishes to compare two conditions for relative abundance of a second protein (Y), which the researcher knows is the direct binding partner of X. First, the crosslinker is reacted in isolation (and in subdued light) with X; the crosslinker attaches at its NHS-ester end to surface primary amines of X, labeling X with several ready-to-activate aryl azide groups. After desalting X to remove non-reacted crosslinker, X is added to samples of Y that represent different treatment conditions (e.g., cell lysates prepared from cells grown in different conditions). Finally, once sufficient time has passed for X and Y to bind one another, the sample is irradiated with UV light to activate the aryl azide moiety, which then conjugates to any protein functional group it is near.
Where nearby amino acids are those of the binding partner (Y), covalent crosslinks between X and Y will form. At this point, the results can be analyzed in several ways. Assuming that the researcher has a specific antibody to detect X, the products can be analyzed by electrophoresis and western blotting. Conjugated proteins will run as one larger protein rather than separate individual proteins, and this difference could be detected and quantified.
Depending on the spacer length and cleavability features of the crosslinker, different particular pairs of protein interactors can be more or less effectively conjugated and analyzed in different ways. Aryl azide compounds that are heterobifunctional with amine-reactive, sulfhydryl-reactive and carbonyl-reactive groups are commercially available.
2. Label protein interactions
Label-transfer is an extension of the heterobifunctional crosslinking just described and is used to investigate protein interactions. Besides the two crosslinking ends, these reagents incorporate a detectable tag or label (e.g., a fluorophore or biotin) and a cleavable spacer arm (usually a disulfide bond).
Sulfo-SBED, a biotin label-transfer reagent. Features of this reagent include the amine-reactive Sulfo-NHS ester (bottom-left), aryl azide group (top-left), biotin (right) and cleavable disulfide bond in the arm of the NHS ester.
Once pairs of interacting proteins have been crosslinked (as described above for hypothetical proteins X and Y), the spacer arm connecting them can be cleaved. This separates the proteins but leaves the label (biotin in the case of Sulfo-SBED) attached to Y. Thus, the biotin label was effectively transferred from the "bait" protein (X) to the "prey" protein (Y).
Diazirine reaction chemistry
Diazirines are a newer class of photo-activatable chemical groups that are being incorporated into various kinds of crosslinking and labeling reagents. The diazirine (azipentanoate) moiety has better photostability than phenyl azide groups, and it is more easily and efficiently activated with long-wave UV light (330 to 370 nm).
Photo-activation of diazirine creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent.
Diazirine reaction scheme for light-activated photochemical conjugation. R represents a labeling reagent or one end of a crosslinker having the diazirine reactive group; (P) represents a protein or other molecule that contains nucleophilic or active hydrogen groups R'.
Although aryl azide reagents are more widely cited in the literature, this is likely to change as diazirine reagents become more widely available and gradually replace aryl azide in most applications.
1. Capture protein interactions
Where diazirine equivalents of heterobifunctional aryl azide reagents are available, all of the same kinds of protein interaction experiments are possible. Currently, several varieties diazirine compounds are available that have an amine-reactive NHS ester at the opposite end.
Example light-activated conjugation with a diazirine crosslinker. SDA is an NHS ester and diazirine heterobifunctional crosslinker. Primary amines of one protein can be labeled in the dark. Then this protein can be added to a complex solution (e.g., cell lysate) and allowed to bind with its specific interactors. Finally, exposure to UV light initiates conjugation to any nearby chemical groups (i.e., the binding partner).
2. Metabolic labeling and crosslinking
The stability and very small size of the diazirine group also enables crosslinking experiments that involve metabolic labeling. For example, Photo-L-Leucine and Photo-L-Methionine are analogs of native amino acids that contain the diazirine group in their side chains. When these compounds are added to culture media instead of their native counterparts, protein synthesis machinery will use the photoreactive versions to build proteins. In this way, proteins themselves become the crosslinking reagents for in vivo crosslinking strategies.
Structures of photoreactive amino acids. These diazirine analogs of leucine and methionine can be incorporated by translation machinery into protein structures as a form of metabolic labeling for protein interaction analysis.
- Covalent crosslinking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts. Wood, C.L. and O’Dorisio, M.S. J Biol Chem (1985) 260:1243-1247
- Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. Gomes, A.F. and Gozzo, F.C. J Mass Spectrom (2010) 45:892-9
- Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Krieg, U.C., Walter, P. and Johnson, A.E.
Proc Natl Acad Sci USA (1986) 83:8604-8606
For Research Use Only. Not for use in diagnostic procedures.