Use Of Bioluminescence Research Articles

Mesopelagic Fish Traits: Functions and Trade‐Offs

ABSTRACTFishes inhabiting the mesopelagic zone of the world's oceans are estimated to account for the majority of the world's fish biomass. They have recently attracted new attention because they are part of the biological carbon pump and have been reconsidered as a contribution to food security. Hence, there is an urgent need to understand how environmental conditions and species interactions shape their assemblages, and how they contribute to the functioning of marine ecosystems. Trait‐based approaches are valuable for addressing these types of questions. However, the biology and ecology of mesopelagic fishes are understudied compared to fishes in shallow and epipelagic waters. Here, we synthesise existing knowledge of traits of mesopelagic fishes and relate them to their role in survival, feeding and growth and reproduction, the key functions that contribute to fitness. Vertical migrations, specialised vision and the use of bioluminescence are among the most striking adaptations to the conditions in the mesopelagic realm. Many traits are interrelated as a result of trade‐offs, which may help to understand selection pressures. While morphological traits are straightforward to observe, major knowledge gaps exist for traits that require frequent sampling, assessment under experimental conditions or age determination. The unique adaptations of mesopelagic fishes need to be included in management strategies as well as fundamental research of the habitat.

Bioluminescence as a functional tool for visualizing and controlling neuronal activity in vivo.

The use of bioluminescence as a reporter for physiology in neuroscience is as old as the discovery of the calcium-dependent photon emission of aequorin. Over the years, luciferases have been largely replaced by fluorescent reporters, but recently, the field has seen a renaissance of bioluminescent probes, catalyzed by unique developments in imaging technology, bioengineering, and biochemistry to produce luciferases with previously unseen colors and intensity. This is not surprising as the advantages of bioluminescence make luciferases very attractive for noninvasive, longitudinal in vivo observations without the need of an excitation light source. Here, we review how the development of dedicated and specific sensor-luciferases afforded, among others, transcranial imaging of calcium and neurotransmitters, or cellular metabolites and physical quantities such as forces and membrane voltage. Further, the increased versatility and light output of luciferases have paved the way for a new field of functional bioluminescence optogenetics, in which the photon emission of the luciferase is coupled to the gating of a photosensor, e.g., a channelrhodopsin and we review how they have been successfully used to engineer synthetic neuronal connections. Finally, we provide a primer to consider important factors in setting up functional bioluminescence experiments, with a particular focus on the genetic model Caenorhabditis elegans, and discuss the leading challenges that the field needs to overcome to regain a competitive advantage over fluorescence modalities. Together, our paper caters to experienced users of bioluminescence as well as novices who would like to experience the advantages of luciferases in their own hand.

The Emerging Use of Bioluminescence in the Lives of Humans

The Emerging Use of Bioluminescence in the Lives of Humans

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display.

Background: Extracellular vesicles (EVs) are attracting interest as a new class of drug delivery vehicles due to their intrinsic nature of biomolecular transport in the body. We previously demonstrated that EV surface modification with tissue-specific molecules accomplished targeted EV-mediated DNA delivery. Methods: Here, we describe reliable methods for (i) generating EGFR tumor-targeting EVs via the display of high-affinity monobodies and (ii) in vitro measurement of EV binding using fluorescence and bioluminescence labeling. Monobodies are a well-suited class of small (10 kDa) non-antibody scaffolds derived from the human fibronectin type III (FN3) domain. Results: The recombinant protein consists of the EGFR-targeting monobody fused to the EV-binding domain of lactadherin (C1C2), enabling the monobody displayed on the surface of the EVs. In addition, the use of bioluminescence or fluorescence molecules on the EV surface allows for the assessment of EV binding to the target cells. Conclusions: In this paper, we describe methods of EV engineering to generate targeted delivery vehicles using monobodies that will have diverse applications to furnish future EV therapeutic development, including qualitative and quantitative in vitro evaluation for their binding capacity.

Use of Bioluminescence for Monitoring Brown Coal Mine Waters from Deep and Surface Drainage

Open-pit mines can cause environmental changes, such as alterations of landscape structure, hydrology, air quality, and river sediments; they can also generate cones of depression. We propose a new method for surveys of mine waters using the example of an open-pit mine in central Poland. This study examines the correlations between bioluminescence and the color of brown coal mine waters and tests whether values of the three-color coordinate system reflected the physicochemical quality of mine waters measured in real-time and in the field. Our results show that alkalinity, pH reaction, and conductivity are higher in surface drainage, while values of trophic parameters (soluble reactive phosphates, total phosphorus, nitrates) are greater in samples representing subsurface drainage. Correlation analysis of bioluminescence with mine water quality parameters showed that only water color had a strong association with bioluminescence. This correlation is stronger for surface drainage, than for mine waters from subsurface drainage. Direct measurement of bioluminescence, resulting from adenosine 5`-triphosphate (ATP) using a luminometer, is a fast and reliable method for evaluation of the characteristics of mine waters in real-time.

From Quorum Sensing to Positional Sensing

With Quorum Sensing (QS) bacteria adapt their physiology and metabolism when they reach a certain density and activate genes relevant for their nutrient metabolism, survival and virulence. Genes controlled by QS have relevance for the pathogenic behavior of bacteria including adherence, secretion, toxin production, immune subversion, biofilm formation and resistance to both antimicrobial and biological/chemical stressors. Most studies assess QS by growing bacteria to various densities in liquid culture. Here, we report that when bacteria are grown on agar plates at various densities, in addition to QS, positional sensing (PS) comes into play. Genes controlled by PS can be studied by cloning their promoter in front of an autonomous bioluminescent cassette (luxCDABE) and measuring their time‐ and position‐dependent expression in biofilms by using an in vivo imaging system (IVIS). The Pseudomonas putida XylR/XylS intergenic region controlling genes relevant for the metabolism and degradation of xylene and related organic molecules was cloned in front of the autonomous luciferase cassette from Photorhabdus luminescence, with the sigma 54 controlled Ps1 promoter regulating the a luxCDABE genes and the Pr promoter regulating the XylR gene. Interestingly, when E. coli carrying this construct were grown to various density on agar plates, bioluminescence was not only expressed in a density‐ and time‐dependent, but also position‐dependent manner. Such positional cues may be mediated by signaling molecules in response to cell density, growth stage, and nutrient availability. Overexpression of the sigma factor 54 (NtrA) from Pseudomonas putida modified the density‐ and time‐dependent response suggesting a regulatory role in QS and PS. The use of bioluminescence in QS and PS promises to reveal important regulatory circuits and communication systems relevant for bacterial normal physiology, pathogenicity and virulence.

An evaluation of Lux technology as an alternative methodology to determine growth rates of Listeria in laboratory media and complex food matrices

Listeria monocytogenes is a foodborne pathogen which is a significant challenge in food production, particularly for ready-to-eat (RTE) products. Incidence of Listeria in food can be reduced by the application of multiple preservative factors or “hurdles”, which include acids, water activity and salts. Studying the growth of Listeria in complex foods is often reliant on laborious plate-counting techniques, therefore alternative methodologies are required. In this study we investigated the use of bioluminescence produced by chromosomally integrated genes encoding luciferase and its substrate to determine microbial growth rates in media and complex food matrices. Five Listeria innocua strains, used as a non-pathogenic surrogate for L. monocytogenes, were transformed with plasmid pPL2luxPhelp, resulting in a collection of Lux-tagged strains. Three test matrices (BHI broth, zucchini purée and béarnaise sauce) were adjusted, testing various combinations of pH (4.7–5.3), water activity (0.96 & 0.98) and undissociated acetic and propionic acid concentrations (0-2 mM). Adjusted matrices were inoculated with a cocktail of Lux-tagged strains and growth monitored over time by both bioluminescence and viable plate counts. Specific growth rates were calculated using both the bioluminescence and plate count data and compared. Statistical analysis showed that specific growth rates determined by bioluminescence did not significantly differ from those determined by plate counts (t-test, P > 0.05), while measurements of bias and accuracy showed good agreement between growth rates determined by both methods. Although plate counts remain the method of choice for detection of very low specific growth rates, this study demonstrates the potential of bioluminescence as a rapid alternative to plate counts for determining microbial growth rates in complex foods.

Bioluminescent flashes drive nighttime schooling behavior and synchronized swimming dynamics in flashlight fish

Schooling fishes, like flocking birds and swarming insects, display remarkable behavioral coordination. While over 25% of fish species exhibit schooling behavior, nighttime schooling has rarely been observed or reported. This is due to vision being the primary modality for schooling, which is corroborated by the fact that most fish schools disperse at critically low light levels. Here we report on a large aggregation of the bioluminescent flashlight fish Anomalops katoptron that exhibited nighttime schooling behavior during multiple moon phases, including the new moon. Data were recorded with a suite of low-light imaging devices, including a high-speed, high-resolution scientific complementary metal-oxide-semiconductor (sCMOS) camera. Image analysis revealed nighttime schooling using synchronized bioluminescent flashing displays, and demonstrated that school motion synchrony exhibits correlation with relative swim speed. A computer model of flashlight fish schooling behavior shows that only a small percentage of individuals need to exhibit bioluminescence in order for school cohesion to be maintained. Flashlight fish schooling is unique among fishes, in that bioluminescence enables schooling in conditions of no ambient light. In addition, some members can still partake in the school while not actively exhibiting their bioluminescence. Image analysis of our field data and model demonstrate that if a small percentage of fish become motivated to change direction, the rest of the school follows. The use of bioluminescence by flashlight fish to enable schooling in shallow water adds an additional ecological application to bioluminescence and suggests that schooling behavior in mesopelagic bioluminescent fishes may be also mediated by luminescent displays.

Hedgehog signaling dynamics in mouse embryos determined by a bioluminiscent reporter.

Determination of cellular signaling in live embryos is key to understand the molecular processes that drive development. Here, we show that a transgenic mouse line carrying a luciferase-based gene reporter of Gli-mediated transcriptional activation (Gli-Luc) displays sonic hedgehog (Shh) signaling in discrete developmental processes during short-term cultures of whole embryos or embryo explants. The bioluminescence in E9.5 embryos was detected in regions in which Shh activity has been demonstrated. Later, in E10.5 embryos, bioluminescence intensity markedly increased, mostly corresponding to the high Shh activity of the developing midbrain and limb. Notably, the dynamic range of the Gli-Luc reporter in the developing limb revealed the progressive emergence of bioluminescence in the zone of polarizing activity, where reporter activity locally increased and spatially spread in agreement with the signaling gradient expected for Shh. In the midbrain of E9.5 mouse embryos, bioluminescence was not detected along the ventral region as expected but, instead, Shh-dependent anterior and posterior bioluminescence foci emerged by E10.5 indicating that the Gli-Luc reporter can only respond transcriptionally to relatively high levels of GliA and/or without the interaction with other transcription factors. The present work supports the use of bioluminescence to identify and study the dynamics of centers of morphogen signaling during mouse embryogenesis.

Bioluminescent Annexin Fusion Proteins (AFPs) for Atherosclerosis Detection

The process of controlled cell death known as apoptosis plays a critical role in the homeostatic tissue maintenance of multicellular organisms under normal, healthy conditions. However, this process (and its dysregulation) has also been implicated in a number of pathologies such as cancer, neurodegenerative diseases, autoimmune diseases, and cardiovascular disease (CVD). This broad involvement of apoptosis in these conditions has led to a sizable amount of research focused on techniques capable of selectively detecting apoptotic cells. Many such techniques rely on the exposure of the specific marker phosphatidylserine (PS) on the surface of apoptotic cells. The protein Annexin V has been shown to selectively bind to exposed PS in a specific manner with high affinity. Detection of apoptosis using this specific interaction has previously been performed utilizing fluorescence or isotopic radio-labeling. In contrast to these approaches, the use of bioluminescence is proven to be an effective means of signal generation in vivo, and provides a number of benefits over both fluorescence and isotopic labeling. Detection via bioluminescence has been shown to exhibit lower background and higher signal-to-noise ratios when compared to fluorescence, and does not carry restrictive handling and disposal regulations encountered when utilizing isotopic labeling. We have begun the development of a library of fusion proteins incorporating both Annexin V and bioluminescent reporters, providing the capability of targeting and binding apoptotic cells as well as generating a detectable bioluminescent signal. The first AFP, containing the bioluminescent reporter Renilla luciferase (RLuc8), was successfully utilized in the detection of atherosclerosis both in vitro as well as in vivo, and was shown to have potential in identifying atherosclerotic plaques associated with CVD). We are now expanding the AFP library to generate a set of unique and tunable AFPs combining Annexin V with a variety of robust bioluminescent reporters. These AFP reporters include: Gaussia luciferase (GLuc), one of the smallest and brightest known luciferases; Aequorin, previously utilized for site-selective and random mutagenesis by our lab resulting in an extensive library of reporters with tunable emission wavelengths and kinetics; and a truncated Vargula luciferase variant (tVLuc), exhibiting glow-type emission kinetics and first reported expression in a bacterial system by our lab. Support or Funding Information This work was supported in part by grants from the National Institutes of Health (1R01GM114321 and R01GM047915). Dr. Sylvia Daunert is grateful to the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in the Department of Biochemistry and Molecular Biology. Apoptosis can be initiated via extrinsic (blue box) or intrinsic (orange box) pathways. However, both pathways converge at the activation of caspases which, in turn, lead to the exposure of phosphatidylserine (PS) on the surface of the apoptotic cell. Under apoptotic conditions, the Annexin Fusion Proteins (AFPs) can bind exposed PS and generate a bioluminescent signal in the presence of their luminescent substrate. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non-invasive tracking of injected bone marrow mononuclear cells to injury and implanted biomaterials.

Tracking the dynamic behaviour of transplanted bone-marrow mononuclear cells (BM-MNCs) is a long-standing research goal. Conventional methods involving contrast and tracer agents interfere with cellular function while also yielding false signals. The use of bioluminescence addresses these shortcomings while allowing for real-time non-invasive tracking in vivo. Given the failures of transplanted BM-MNCs to engraft into injured tissue, biomaterial scaffolds capable of attracting and enhancing BM-MNC engraftment at sites of injury are highly sought in numerous tissue engineering applications. To this end, the results from this study demonstrate a new longitudinal tracking model that can non-invasively determine exogenous BM-MNC homing and engraftment to biomaterials, providing a valuable tool to inform the design of scaffolds with implications for countless tissue engineering applications.

In Vivo Tracking of Streptococcal Infections of Subcutaneous Origin in a Murine Model.

Generation of plasmin in vivo by Streptococcus pyogenes is thought to localize the active protease complexes to the pathogen surface to aid in tissue dissemination. Here, we chose to follow cutaneous streptococcal infections by the use of non-invasive bioluminescence imaging to determine if this pathogen can be followed by this approach and the extent of bacterial spread in the absence of canonical plasminogen activation by streptokinase. Mice were injected subcutaneously with either bioluminescent strains of streptococci, namely Xen20 and Xen10 or S. pyogenes ALAB49. Bioluminescence imaging was performed daily and results were correlated with microbiological and histological analyses. Comparative analysis of chronologic non-invasive datasets indicated that Xen20 did not disseminate from the initial infection site. Contrary to this, microbiological and histological analyses of Xen20 mice for total bacterial burden indicated sepsis and widespread pathogen involvement. The use of bioluminescence in microbe-based studies requires genomic and pathologic characterization to correlate imaging results with underlying pathology.

Host preference, site fidelity, and homing behavior of the symbiotically luminous cardinalfish, Siphamia tubifer (Perciformes: Apogonidae)

The sea urchin cardinalfish, Siphamia tubifer (Perciformes: Apogonidae), is unusual among coral reef fishes for its use of bioluminescence, produced by symbiotic bacteria, while foraging at night. As a foundation for understanding the relationship between the symbiosis and the ecology of the fish, this study examined the diel behavior, host urchin preference, site fidelity, and homing of S. tubifer in June and July of 2012 and 2013 at reefs near Sesoko Island, Okinawa, Japan (26°38′N, 127°52′E). After foraging, S. tubifer aggregated in groups among the spines of the longspine sea urchin, Diadema setosum, and the banded sea urchin, Echinothrix calamaris. A preference for D. setosum was evident (P 25 mm standard length, P < 0.01), and choice experiments demonstrated the ability of S. tubifer to recognize and orient to a host urchin and to conspecifics. Tagging studies revealed that S. tubifer exhibits daily fidelity to a host urchin; 43–50 and 26–37 % of tagged individuals were associated with the same urchin after 3 and 7 days. Tagged fish also returned to their site of origin after displacement; by day two, 23–43 and 27–33 % of tagged individuals returned from displacement distances of 1 and 2 km. These results suggest that S. tubifer uses various environmental cues for homing and site fidelity; similar behaviors and cues might be used by larvae for recruitment to settlement sites and for the acquisition of luminous symbiotic bacteria.

&lt;p&gt;&lt;strong&gt;Study of the luminescence in the black brittle-star Ophiocomina nigra: toward a new pattern of light emission in ophiuroids*&lt;/strong&gt;&lt;/p&gt;

The black brittle star Ophiocomina nigra, common in the English Channel, is known to produce mucus when attacked. This mucus, already known for its antifouling capabilities and its role in the feeding and the locomotion behaviours of the brittle star, also emits weak light. We describe and characterize this emission of bioluminescence, thanks to a chemical triggering by hydrogen peroxide. It appears that the light emitted is 1000 times less intense than the light emitted by other brittle star species (Ophiopsila aranea and Amphipholis squamata). The luminous capabilities are homogeneously spread along the arms of the brittle star, what goes against the use of bioluminescence as a sacrificial lure. The mechanical stimu­lation of arms before chemical triggering strongly enhances the luminous capabilities of the brittle star. Luminous mucus emission can be associated with other defensive function, such as a smoke screen effect or a burglar alarm, but these two functions require intense light emissions. The fact that the luminous component is excreted outside the body might be in favour of the aposematic use of light, i.e., as a signal to warn predators of the toxicity or unpalatability of its prey.

Thoughts on the diversity of convergent evolution of bioluminescence on earth

AbstractThe widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms.Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms.Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

Spontaneous flash communication of females in an Asian firefly

Fireflies are well known for the use of bioluminescence for sexual communication. In species using flash signals for pair formation, species and sexual identity are conferred by flash timing parameters such as flash duration, flash interval, flash number, and response delay. In dialog fireflies in North America, the male is the advertiser and the female is the responder. In these species, the male flash signal parameter varies depending on species, but the female flash signal parameter is limited only to response delay. However, in fireflies other than dialog fireflies, sexual flash communication is not well studied. Although many female-advertisement-like fireflies are reported, we have no confirmed case of sexual communication in a female-advertisement species. Here, we report the sexual flash communication of an Asian firefly, Luciola (Hotaria) parvula, in which the female flashes spontaneously. By using an electronic firefly, we confirm experimentally that males are specifically attracted to flashes with a female-specific flash duration. This is the first experimental report of sexual communication of a female advertiser in firefly communication. In this species, females call males usually with spontaneous flashes unlike dialog fireflies.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

On the use of bioluminescence for estimating shear stresses over a rippled seabed

The bioluminescence emitted by micro-planktonic organisms has been adopted to detect shear stresses within oscillating flows over small scale bedforms. To this aim, an experimental campaign has been planned in order to optimize optical detectors and to address peculiar needs of biological organisms. Oscillating flow over ripples has been simulated in an annular cell, an apparatus that permits one to carry out experiments on ripple formation under well controlled conditions. Bioluminescence emission within the annular cell ascertains the existence of a high shear stress regime induced by the presence of the rippled bed in the range of Reynolds numbers between 2000 and 11000. Stress maps obtained from the experimental campaign showed the stronger stresses close to the rippled bed, with maximum values ranging between 15 and 35 Pa. 2D numerical simulations of oscillating flows over a rippled bed have been also run reproducing the experimental conditions, showing a good agreement on the position of the high stress regions, but underpredicting the maximum shear stress. Bioluminescence appears to be a valid instrument to characterize the flow and in particular to visualize high stress areas, leading to the possibility to measure shear stresses directly. Though limits occur, mostly due to the difficulties of dealing with biological organisms, such an approach can be considered as a starting point for the development of a bio-inspired visually based system for flow measurements.

Optimisation of Bioluminescent Reporters for Use with Mycobacteria

Background Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.Methodology/Principal FindingsOne of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.Conclusions/SignificanceWhile much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Optimization of luminescent assay for screening of cyclin-dependent kinase 2 inhibitors

Cyclin-dependent kinases are most extensively studied targets for cancer chemotherapy since the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised. Cyclin-dependent kinases ensure the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Deregulation of the cyclin-dependent kinase 2 activity by cellular and external factors leads to many diseases like cancers. Different methods like radiolabeled, fluorescence and luminescence are available for screening of library of compounds against kinases. However, bioluminescent methods offer several advantages like low background and no effect of fluorescent compound interference. Present study is focused on development, optimization and validation of cyclin-dependent kinase 2 assay which is suitable for identification potent and selective, ATP competitive and non-competitive inhibitors of cyclin-dependent kinase 2. The aim of present investigation was to optimize the assay for cyclin-dependent kinase 2/cylin A and cyclin-dependent kinase 2/cyclin E with use of bioluminescence based biochemical reaction. Both cyclin-dependent kinase 2 which are cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E complexes, have different affinity for ATP. Therefore, both isoform analogs of cyclin-dependent kinase 2 were optimized separately. Optimum cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E concentration were found to be 250 ng/well and 200 ng/well, respectively. Optimum substrate (histone H1) concentration was found to be 2.5 mg/ml for both cyclin-dependent kinase 2 analogs. Optimum reaction time was found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes.