How to Choose A Firefly Luciferin Substrate

by Katharine Martin

With the different types of substrates out there, how do you choose which luciferin substrate to use for your experiment?

For the most common bioluminescence work, you’ll typically want to choose either D-luciferin sodium salt or D-luciferin potassium salt. However, there are some other factors to consider when choosing, and we’ll dive into that in this article.

Just a quick note about this article, we’re only focusing on the different firefly luciferin substrates. We won’t be talking about choosing a coelenterazine or whether to choose coelenterazine or luciferin for your experiment. To find out more about that, you might want to check out our article: ”How Different Luciferin-Luciferase Systems Are Used In Biotechnology.”

Another great article to check out when deciding about luciferin is “10 Things and Beyond to Consider When Shopping or Using Luciferin.” This article goes into detail about solubility considerations, assay considerations, quality, cost and so much more.

How to choose between D-luciferin potassium salt vs. D-luciferin sodium salt

So how do you choose between D-luciferin potassium salt and D-luciferin sodium salt when both are so commonly used?

There are three common reasons to choose one versus the other.

Solubility: D-luciferin sodium salt has better solubility in water (up to 100 mg/ml) compared to D-luciferin potassium salt (55 – 60 mg/ml). However, if you’re making a 15 mg/ml stock solution, either substrate will be just fine.
Stability: D-luciferin potassium salt has a little bit better stability than D-luciferin sodium salt.
Protocol: If stability and solubility aren’t as essential to your project, sometimes it boils down to the protocol or papers you’re referencing.

Why would you use L-luciferin?

The isomer L-luciferin is known as an impurity to D-luciferin. So why would you ever use L-luciferin in a bioluminescent experiment?

One answer is that L-luciferin can act as a competitive inhibitor of D-luciferin, increasing the apparent Km for D-luciferin. While this property has been studied, its practical application in dual bioluminescent assays requires further verification (Thouand, G., & Marks, R., 2014).

Why would you use DMNPE-caged luciferin?

One of the advantages of DMNPE-caged luciferin is that it can cross the cell membrane (it’s more permeable). Researchers sometimes select this luciferin to use to measure intracellular functions (inside the cell).

Therefore, in certain situations it can be a great choice for in vivo settings. But then you might wonder, “couldn’t I just use D-luciferin sodium or potassium salt for that?” At physiological pH, D-luciferin, being negatively charged, may have reduced cell membrane permeability compared to neutral derivatives. pH adjustment can potentially improve permeability, though this approach has limitations in biological systems. Therefore, DMNPE-caged luciferin can be a better alternative.

Another obstacle is when dealing with the cell wall. The negative charge of a bacterial cell wall will oppose the negative charge of D-luciferin in a neutral pH. Again, one way to overcome this is by carefully lowering the pH. But using DMNPE-caged luciferin can make this easier.

These caged luciferins are permeable derivatives. Once it’s in the cell, it can be converted into its active form either through an enzymatic process or through photoactivation (Brovko, 2010).

how DMNPE-caged luciferin penetrates the bacterial cell wall. DMNPE-caged luciferin is a neutral derivative, whereas d-luciferin is negatively charged. The cell wall is also negatively charged, causing permeability issues for d-luciferin but not for caged-luciferin

Figure 1. Displays the concept behind caged-luciferin for bacterial cells. The bacterial cell wall is negatively charged, and D-luciferin sodium and potassium salt are also negatively charged. However caged luciferin derivative substrates are neutral and can therefore penetrate the negatively charged bacterial cell wall.

Why would you use click beetle luciferin?

Whether from the families of Elateridea (click beetle), Phengodidae (railroad worms) and Lampyridae (fireflies), the luciferase reaction is the same. So when would you choose to use click beetle luciferin?

The reason has to do with max emission or color emission. Within the click beetles, there are some species and derivatives capable of a higher wavelength.

Signal production within the blue range of the spectrum is still within range of high tissue absorption. For deep tissue imaging, this starts to pose an obstacle. This is why having a higher emission luciferin such as click beetle can offer some advantage (Sônego et al., 2019).

One thing to keep in mind, is there are also modified firefly luciferin substrates that are red shifted.

where different luciferin-luciferase systems are placed on the visible light spectrum based on max wavelength emission and species type - species include firefly, renilla, cypridina, guassia, and click beetle

Figure 2. Shows the max emission for different bioluminescent substrates from different organisms. D-luciferin emits a max wavelength of roughly 560 nm. Certain click beetles and mutant click beetles can emit a higher wavelength (613 nm) in the orange-red region of the visible spectrum (Brovko, 2010, pp. 8).

How to get started with luciferin

1. The first step is simply knowing what you’re studying and the experimental assay you’re going to run. Are you doing a dual-luciferase assay? Are you working in vivo or in vitro? There are several different assays and experiments that use luciferin.

Part of determining your approach comes down to what you’re studying. For instance, are you studying:

· Protein-protein interactions

· Cell viability

· Pathogenesis

· Gene expression

· Gene regulation and function

2. Once you know the assay you’re running, look at papers and protocols that are closely related to your project. Here you will find the type of luciferin used, conditions, amounts used, what sample was being used, possible information about vectors, etc.

3. Look at vendor protocols for information about stock solutions, optimal buffers, and more. Your vendor will have a lot of information about how to dissolve your luciferin, and will potentially have information about proper buffers to use and how to optimize.

For instance, GoldBio provides stock solution protocols for its luciferins, as well as solution storage information. Additionally, our Luciferin In Vitro Handbook has detailed information on creating a luciferase assay buffer as well as how to dissolve and use Luciferin. And our Luciferin In Vivo Handbook not only has buffer recommendations but also has information on substrate injection.

There are all kinds of luciferin modifications and derivatives available, and it can seem pretty daunting. Really, the best way to approach choosing a luciferin is identifying what your experimental goals are, then evaluating the different specifications of each available luciferin. Doing so will help you quickly narrow down your choices until you’re confident in your selection.

GoldBio Luciferin

D-Luciferin Potassium Salt

D-Luciferin Sodium Salt

L-Luciferin

DMNPE-caged Luciferin

References

Brovko, L. (2010). Bioluminescence and fluorescence for in vivo imaging: In vivo optical imaging. Bellingham, WA: SPIE Press.

Thouand, G., & Marks, R. (Eds.). (2014). Bioluminescence: Fundamentals and Applications in Biotechnology-Volume 2. Springer Berlin Heidelberg.

Sônego, F., Bouccara, S., Pons, T., Lequeux, N., Danckaert, A., Tinevez, J. Y., ... & Tournebize, R. (2019). Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence. Frontiers in bioengineering and biotechnology, 7, 73.

Thorne, N., Inglese, J., & Auld, D. S. (2010). Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chemistry & Biology, 17(6), 646-657.