ACT assay
Actinorhodin Assay
Overview
Actinorhodin is a pigmented secondary metabolite produced by Streptomyces coelicolor and related species. In alkaline conditions, actinorhodin converts into a blue chromophore with a characteristic absorbance peak near 633 nm. Its concentration can be determined spectrophotometrically using the Beer–Lambert law with a reported molar extinction coefficient.
Principle
- Equal volumes of culture supernatant and strong base (e.g. NaOH) are combined.
- Under alkaline conditions, actinorhodin develops a blue colour measurable at 633 nm.
- Clarified samples are read spectrophotometrically, and concentration is calculated.
- Specific production yields can be expressed as µM actinorhodin per mg cell dry weight (CDW).
Example Assay Description
Equal volumes of culture supernatant and 1 M NaOH were mixed by vortexing. The samples were centrifuged at 1000 × g for 5 min, and the absorbance at 633 nm was measured.
The molar extinction coefficient for actinorhodin is: ε = 15,135 M⁻¹ cm⁻¹
Using the Beer–Lambert law (A = ε × l × c), the concentration was calculated. The specific production yield was expressed as µM actinorhodin per mg of cell dry weight (CDW).
Notes
- Always report the extinction coefficient, wavelength, and pathlength used.
- Use appropriate blanks (e.g. medium + base).
- Keep absorbance within the linear range of the spectrophotometer.
- Related “blue pigments” may contribute to absorbance signals; confirm specificity where necessary.
- Consistency in reporting wavelength and extinction coefficient is essential for comparability between studies.
References
- Hobbs, G., Frazer, C. M., Gardner, D. C. J., Flett, F., & Oliver, S. G. (1990). Pigmented antibiotic production by Streptomyces coelicolor A3(2): kinetics and the influence of nutrients. Microbiology, 136(11), 2291–2296. DOI
- Kang, S. G., Jin, W., Bibb, M., & Lee, K. J. (1998). Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor grown in continuous culture. FEMS Microbiology Letters, 168(2), 221–226. DOI
- Bystrykh, L. V., et al. (1996). Production of actinorhodin-related “blue pigments” by Streptomyces coelicolor A3(2). Journal of Bacteriology, 178(8), 2238–2244. DOI
