Firefly enzyme can be used as an indicator of intracellular pH

The application was suggested by Brazilian researchers in an article published in Photochemical & Photobiological Sciences.

By Noêmia Lopes

Agência FAPESP – In an article published by the journal Photochemical & Photobiological Sciences, researchers at the Sorocaba campus of the Federal University of São Carlos (UFSCar) in São Paulo State, Brazil, demonstrated the possibility of using luciferase enzymes from fireflies as indicators of intracellular pH in bacteria – a novel application of luciferase.

The demonstration of this application was achieved in the course of doctoral research performed by Gabriele Verônica de Mello Gabriel in UFSCar’s evolutionary genetics and molecular biology graduate program under the supervision of Vadim Viviani, head of the university’s Bioluminescent Systems Biochemistry & Biotechnology Laboratory.

Mello Gabriel first worked with firefly luciferase for her master’s research, when she was a student in the biotechnology and environmental monitoring graduate program, evaluating the possibility of using luciferase from Macrolampis sp2, which is found in the Atlantic rainforest, as an indicator of intracellular pH in bacteria. Viviani applied for a patent on this application in 2005.

“We already knew that firefly luciferase bioluminescence shifts from yellow-green to red under acidic pH, at high temperatures or in the presence of heavy metals.

Now we’ve shown that the red-to-green light-intensity ratio can be effectively used to indicate intracellular pH in bacteria,” Viviani told Agência FAPESP.

According to the researcher, this advance paves the way for the use of Macrolampis sp2 luciferase as an indicator of pH in other cells, such as those of mammals.

This could be useful, in turn, as a method for monitoring the changes in pH associated with pathological biological processes such as carcinogenesis, inflammation, acidosis and apoptosis.

This is a new type of application for luciferases, which have been used for decades as luminous markers of gene expression in cells.

“The additional advantage of our recent discovery is that it enables the use of a single luciferase gene to monitor more than one intracellular physiological event. For example, it could be deployed to analyze the energy states of cells – which are calculated based on light intensity, which is proportional to intracellular adenosine triphosphate [ATP] content – and, at the same time, their acidity using spectral parameters, i.e., light color shifts,” Viviani said. “It’s no accident that cell death is accompanied by falling levels of ATP and acidification.”

Continuing her doctoral research, Mello Gabriel will now investigate the applicability of Macrolampis sp2 luciferase in mammalian cells, in partnership with researchers at the National Institute of Advanced Industrial Science & Technology (AIST) in Tsukuba, Japan.

“Here in our lab, the next step will be to evaluate the use of the enzyme as an intracellular biosensor for heavy metals, such as copper, zinc and mercury, to detect the presence of toxicity in water,” Viviani said.

According to the researcher, the plan is to develop a center for biophotonic applications of luciferases and bioluminescence for environmental and biomedical applications.

Bioluminescence colors

In another article, published in July 2014 in Biochemistry, UFSCar’s researchers, in partnership with collaborators at the University of Electro-Communications (UEC) in Tokyo, Japan, explored the mechanism that determines bioluminescence colors in beetle luciferases.

Bioluminescence in fireflies and other beetles also ranges from green to red, employing the same biochemical reaction with the same reagents: luciferin (a fluorescent pigment that acts as a light emitter when oxidized), energy stored in ATP molecules, oxygen, and luciferase enzymes.

“The reaction is identical in all species, but small variations in the structures of the luciferase enzymes and the chemical properties of their active sites [the protein cavities in which the chemical reaction that oxidizes luciferin occurs, resulting in the production of light] are responsible for color modulation,” Viviani said.

To achieve a better understanding of these variations, Japanese researcher Takashi Hirano (UEC) synthesized analogs of luciferin – compounds produced from the original substrate but with modifications to some parts of the molecule – that helped to identify the chemical groups in the luciferin molecule that interact with luciferase to modulate bioluminescence colors.

The Brazilian researchers characterized the bioluminescence properties of these analogs that were synthesized in Japan using an extensive repertoire of luciferases, cloned and modified through genetic engineering, from Brazilian species that emit various colors of light (green, yellow-green, blue-green, yellow, orange and red). This repertoire of multicolor luciferases is the product of cloning and genetic engineering experiments that have been conducted for over 15 years by Viviani and his research group.

“Using this combined approach, which involves the modification of both the chemical structure of luciferin and the luciferase active site, we identified the most likely chemical form of the light-emitting molecule [oxyluciferin] and mapped some of the physical and chemical interactions between the luciferase active site and this light-emitting molecule that are responsible for the light’s colors,” Viviani said.

“In addition to helping unlock the mystery behind the colors of firefly bioluminescence, this study provides input for the development of new guidelines for the engineering of luciferase enzymes and combinatory chemistry, allowing us to extend the range of bioluminescence assay colors that are widely used in the biomedical and environmental fields.”

Other participants in the research included Scientific Initiation Scholar Deimison Rodrigues Neves, PhD student Danilo Trabuco do Amaral, and post-doctoral student Rogilene Aparecida Prado.

Amydetes vivianii

Among the cloned luciferases used by the research group at UFSCar for their investigation of the mechanism that determines bioluminescence colors is one from Amydetes vivianii, a species that was described early this year and is named for the researcher Viviani, who discovered it on the Sorocaba campus shortly after the facility was unveiled in 2006.

The species was described by taxonomist Luiz Felipe Lima, a PhD student affiliated with the Entomology Laboratory at the Federal University of Rio de Janeiro (UFRJ). A. vivianii is known for producing intense blue-green light. Viviani and his students cloned its luciferase enzyme in 2011 for use as a reagent in quantifying cellular ATP, serving as a bioluminescent marker and as a biosensor to detect toxic agents.

“The discovery and description of a new firefly species, a target for research that has already resulted in the cloning of various enzymes of biotechnological interest, illustrates the importance and urgency of these studies and of conserving the native biodiversity of Brazilian biomes,” Viviani said. This last project was supported by a regular grant extended under FAPESP’s BIOTA Program.

According to Viviani, the greatest threats to firefly species such as A. vivianii are the disappearance of habitats as a result of urbanization and the increasing prevalence of monoculture as well as growing light pollution in urban areas.

“The worst threat of all is happening not in São Paulo State but in the Center-West region and in the Amazon, where vast areas have been deforested to make way for extensive soya and sugarcane monocultures,” Viviani said.

The researcher warned that in the vicinity of Costa Rica, Mato Grosso do Sul State, dozens of firefly species have disappeared, including two (the railroad worm and the larval click beetle) that had yielded technological products of importance to human health.

“Fireflies and glowworms are important bioindicators of environmental quality,” he said. “Their disappearance not only means a loss of natural beauty but also sounds the alarm for the unwary: our environment is collapsing, and society will lose the benefits it can provide in the form of renewable resources and important products for human and environmental health.”