Acriflavine Hydrochloride

Overview

Acriflavine hydrochloride is an acridine dye with a long history in microbiology and molecular biology. It is an intensely orange powder, soluble in water and ethanol, and it binds strongly to DNA and RNA.

The binding is what makes it useful. The acridine ring system is flat, and it slides between the stacked base pairs of a nucleic acid duplex, a mode of binding called intercalation. Once bound, the dye's fluorescence changes, so it reports on the nucleic acid it has attached to. That single property underlies its three main laboratory roles: as a fluorescent stain for visualising nucleic acids, as a probe in binding and intercalation studies, and as a fluorometric substrate in bacterial efflux-pump assays.

The acridines also occupy a particular place in the history of molecular genetics, having been the compounds used to establish that frameshift mutations arise from insertion and deletion of single bases. Acriflavine remains a reference intercalator in that work.

Key features

• Assay ≥ 90% by HPLC
• Strong, well-characterised intercalative binding to double-stranded DNA
• Soluble in both water and ethanol, so it suits aqueous and part-organic systems
• Fluorescence excitation near 452 nm fits standard blue-light and FITC-range optics
• Established substrate for multidrug efflux-pump activity measurements

Identification

Applications

Frequently Asked Questions

Mode of Action

Acriflavine is a planar tricyclic acridine cation. It binds double-stranded DNA by intercalation, inserting between adjacent stacked base pairs roughly perpendicular to the helical axis, which locally lengthens and unwinds the duplex. Binding is driven by stacking interactions with the bases and by the electrostatic attraction between the cationic dye and the polyanionic backbone, so it is sensitive to ionic strength.

Intercalation accounts for both of the dye's uses. The change in the fluorophore's environment on binding alters its fluorescence, which is the basis of its use as a stain and as a binding probe. Distorting the helix also interferes with replication and transcription, which is the basis of its growth-inhibitory effect on microorganisms and of its classical activity as a frameshift mutagen.

Physical and Spectral Properties

Spectral maxima shift with solvent, pH, ionic strength and on binding to nucleic acid. The figures above are approximate values for the free dye in water and should be confirmed on your own instrument for quantitative work.

Handling Guidance

1. Weigh the powder in a fume hood or ventilated weighing enclosure, wearing chemical safety goggles, nitrile gloves and a fastened laboratory coat.
2. Prepare stock solutions in distilled or deionised water, or in ethanol for less polar systems. Protect them from light from the moment they are made.
3. Store stock and working solutions at 4 °C in amber or foil-wrapped containers. Prepare small volumes so they are used while fresh.
4. Titrate the working concentration empirically for your staining or assay system. Optimal loading varies with sample type, detection method and instrument sensitivity.
5. Minimise illumination time during measurement, and keep exposure consistent across samples, to limit photobleaching.
6. Collect all dye-containing waste, including stained material and rinse water, for disposal as potentially mutagenic hazardous waste. Nothing goes to drain.

Limitations

• The product is a mixture, so the component ratio can vary between batches and suppliers and may affect quantitative results.
• The dye photobleaches, which limits long time-course fluorescence measurements and repeated imaging of the same field.
• Fluorescence is sensitive to pH, ionic strength and binding state, so controls must be run under the sample conditions rather than in buffer alone.
• Intercalating dyes bind nucleic acid non-specifically and do not distinguish sequence or, on their own, DNA from RNA.
• All dye-containing waste requires a hazardous waste route, which adds cost and handling compared with non-intercalating alternatives.

Literature and References

1. Peacocke AR, Skerrett JNH. The interaction of aminoacridines with nucleic acids. Trans Faraday Soc. 1956;52:261–279.
2. Lerman LS. Structural considerations in the interaction of DNA and acridines. J Mol Biol. 1961;3(1):18–30.
3. Crick FHC, Barnett L, Brenner S, Watts-Tobin RJ. General nature of the genetic code for proteins. Nature. 1961;192:1227–1232.
4. Wainwright M. Acridine — a neglected antibacterial chromophore. J Antimicrob Chemother. 2001;47(1):1–13.
5. Lee K, Zhang H, Qian DZ, Rey S, Liu JO, Semenza GL. Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proc Natl Acad Sci USA. 2009;106(42):17910–17915.
6. Blair JMA, Piddock LJV. How to measure export via bacterial multidrug resistance efflux pumps. mBio. 2016;7(4):e00840-16.

References are provided for technical context. Citation of a published study is not a claim about this product's performance or suitability for that application.

Hazard Identification

Precautionary statements: P264 Wash thoroughly after handling. P270 Do not eat, drink or smoke when using this product. P273 Avoid release to the environment. P280 Wear protective gloves, protective clothing and eye protection. P301+P312+P330 IF SWALLOWED: call a poison centre or doctor if you feel unwell; rinse mouth. P305+P351+P338+P310 IF IN EYES: rinse cautiously with water for several minutes, remove contact lenses if present and easy to do, continue rinsing, and immediately call a poison centre or doctor. P332+P313 If skin irritation occurs, get medical advice. P391 Collect spillage. P501 Dispose of contents and container in accordance with local regulations.

Composition

First Aid

Handling, Spills and Disposal

• Handle in a fume hood or ventilated weighing enclosure. Avoid generating dust.
• Do not eat, drink or smoke in the handling area. Wash hands thoroughly after use.
• Store locked up or in a restricted chemical store, segregated from oxidising agents.
• Protect surfaces before opening the container; the dye stains strongly and spreads easily.
• Spills: avoid dust. Collect dry material mechanically or by HEPA vacuum into a labelled closed container, then wash the area and collect the washings as chemical waste. Do not wash a dry spill first, as this spreads it.
• Environmental precautions: prevent entry to drains, waterways and sewers. The product is toxic to aquatic life with long-lasting effects.
• Disposal: dispose of the product, its solutions, contaminated packaging, stained material and rinse water as hazardous chemical waste through a licensed contractor, in accordance with local regulations.

Quality Control Specification

Identity Confirmation

Identity is confirmed by the UV-visible absorption spectrum of an aqueous solution against a reference, with the principal absorption maximum at approximately 452 nm.