Luminescent enzyme substrate

1. Solution Preparation [4]
1.1 Stock Solution Preparation
Solvent: Methanol or ethanol
Concentration: 10 mM (optimize according to experimental conditions).
1.2 Working Solution Preparation
Dilute with PBS or cell culture medium to 10 µM (optimize according to experimental conditions).
Note: Stock and working solutions should be prepared immediately before use and stored protected from light.

2. Cell Staining
2.1 Transfect HeLa cells with BRAC or G5A.
2.2 Add 10 µM Coelenterazine h to the cell plate.
2.3 Incubate the plate with the dye in a cell culture incubator for 1–4 hours (optimize according to experimental conditions).
2.4 Detect by fluorescence microscopy (Ex/Em = 437/466 nm).

Coelenterazine h (1–10 μM) can be used as a luminescence substrate for RLuc8 [4].
In measurements of Ca²⁺ binding kinetics using BRAC, 5 nM BRAC protein was rapidly mixed with 20 μM Coelenterazine h in buffers containing various Ca²⁺ concentrations. The Venus emission intensity (530 nm) of BRAC was then monitored at 1 kHz [4].

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Coelenterazine h (1-10 μM) hydrochloride can be used as a luminescent substrate for RLuc8[4]. In the Ca2+ binding kinetics measurement of BRAC, 5 nM BRAC protein was rapidly mixed with 20 μM Coelenterazine h hydrochloride in different concentrations of Ca2+ buffer, and the Venus (530 nm) emission intensity of BRAC was monitored at 1 kHz[4].

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The drift observed in both RLuc8 and Venus signals might be caused by the uptake and consumption of coelenterazine-h or the change in cell shape.[4]
We then measured the Ca2+-association kinetics of BRAC by stopped-flow photometry system. However, the time course data we obtained was composed of at least two exponential decay components (ι<0.1 sec) which were thought to be derived from both Ca2+ and coelenterazine-h binding to BRAC.[4]

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The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using Coelenterazine H, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin[2].

Protein expression, purification and Ca2+ titration in vitro[4]
Recombinant BRAC protein with N-terminal polyhistidine tags was expressed in Escherichia coli [JM109(DE3)] at 23°C, purified using an Ni-NTA column. Emission spectra of BRAC were measured using a spectrophotometer and a microplate reader. Final concentration of 1–10 µM coelenterazine-h was used as the luminescent substrate for RLuc8. Ca2+ titration was performed by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA), N-(2-Hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), or Nitrilotriacetic acid (NTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentrations were calculated by using 0.15, 4.3 and 170 µM as the K d value of EGTA, EDTA-OH and NTA for Ca2+, respectively [20]. A Ca2+ titration curve was used to calculate apparent K d value by non-linear regression analysis. The averaged data from eight independent measurements were fitted to the Hill equation using Origin7 software.

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Measurement of Ca2+ binding kinetics[4]
Measurements of Ca2+ binding kinetics of BRAC were performed by using stopped-flow photometry system consisting of RX.2000 rapid mixing stopped-flow unit and FP-750 spectrophotometer. Emission intensity of Venus (530 nm) from BRAC were monitored at 1 kHz just after rapid mixing of 5 nM BRAC protein with 20 µM coelenterazine-h in various concentration of Ca2+ buffer. In this experiment, we did not mix coelenterazine-h with BRAC prior to measurement to avoid undesirable consumption of coelenterazine-h by Rluc8 in BRAC during sample preparation. Thus, time course of emission intensity in the stopped-flow experiments consists of three components of kinetics derived from Ca2+ binding to BRAC, coelenterazine-h binding to BRAC, and catalytic oxidation of coelenterazine-h by BRAC. To estimate the catalytic oxidation of coelenterazine-h by Rluc8 in BRAC, we measured time course of emission intensity change after mixing BRAC with 20 µM coelenterazine-h in Ca2+-free solution, and used the obtained data as a “base line”. Then, we measured time course of both association and dissociation of Ca2+ to and from BRAC by mixing 1 volume of BRAC in Ca2+-free buffer with 25 volume of solution containing 1.69 µM Ca2+, and 1 volume of BRAC in 1.69 µM Ca2+ solution with 25 volume of Ca2+-free buffer, respectively. The averaged data from at least 5 independent measurements were used for following analysis. The averaged time course data for association and dissociation kinetics were subtracted by the base line to remove the fraction derived from autonomous catalytic oxidation of coelenterazine-h by BRAC. Then, the time constants (ι) were calculated by means of curve fitting in single exponential equation using the data from 0.2 sec to 2.0 sec to minimize contribution of signal derived from association of coelenterazine-h with BRAC just after mixing. Measurements of Ca2+ binding kinetics of YC3.60 were performed as shown previously. In the stopped-flow experiment, final Ca2+ concentration was controlled by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentration in every solution was confirmed with Ca2+-sensitive electrode which is calibrated with a CaCl2 standard solution.

Cell culture and transfection[4]
Hela cells were cultured in a homemade 35-mm glass-bottom dish in DMEM containing 10% fetal bovine serum. Cells were transfected with plasmids by means of Lipofectamine 2000. At 1 to 2 days after transfection, cells expressing BRAC or G5A were subjected to imaging. 10 µM coelenterazine-h were added to the culture medium just before observation of BRAC and 1–4 hours before observation of G5A.

[1].New bioluminescent coelenterazine derivatives with various C-6 substitutions. Org Biomol Chem. 2017 Aug 23;15(33):7008-7018.

[2].Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins. J Cell Biol. 1993 Apr;121(1):83-90.

[3].Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat Commun. 2016 Dec 14:7:13718.

[4].Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level. PLoS One. 2010 Apr 1;5(4):e9935.

Renin luciferin belongs to the phenolic and imidazopyrazine class of compounds and has the function of luciferin. It is derived from the hydride of imidazo[1,2-a]pyrazine. To expand the bioluminescent substrates, we designed and synthesized a series of novel coelentrin analogs with different substituents at the C-6 position of the imidazopyrazinone core. Some of these analogs have shown superior bioluminescent performance in both in vitro and in vivo biological evaluations compared to DeepBlueC™ or natural coelentrin, making these derivatives one of the ideal substrates for the bioluminescent application of renin luciferin. [1] Luminescent imaging has attracted much attention as a promising bioimaging method when fluorescence imaging is not applicable. However, its wider application in multicolor and dynamic imaging is limited by the lack of bright luminescent proteins that emit across the entire visible spectrum. This paper reports five spectral variants of a novel high-brightness luminescent protein—enhanced nanolamp (eNL). eNL consists of the brightest luciferase NanoLuc tandem with fluorescent proteins of different colors. eNL enables five-color live-cell imaging and can detect single protein complexes or even single molecules. We also developed an eNL-based Ca²⁺ indicator with a signal change of up to 500%, which can be used to image spontaneous Ca²⁺ dynamics in cardiomyocyte and nerve cell models. These eNL probes can not only achieve multicolor imaging of live cells, but also sensitively image a variety of proteins, and can effectively detect them even at very low expression levels. [3] We used efficient bioluminescent resonance energy transfer (BRET) between bioluminescent proteins and fluorescent proteins with high fluorescence quantum yields to enhance the luminescence intensity, thereby achieving near real-time single-cell imaging without external illumination. Methods/Main Findings: We developed a self-luminescent calcium ion (Ca²⁺) indicator, BRAC, using bioluminescent resonance energy transfer (BRET) technology. The indicator consists of the calcium ion-binding protein calmodulin and its target peptide M13, sandwiched between the yellow fluorescent protein variant Venus and the enhanced René luciferase RLuc8. By adjusting the relative dipole orientation of the chromophore of the luminescent protein, the dynamic range of the BRET signal change of BRAC was increased by 60%, which is the largest dynamic range reported to date for BRET-based indicators. Using BRAC, we successfully visualized the dynamic changes of Ca²⁺ at the single-cell level with a time resolution of 1 Hz. In addition, the BRAC signal was acquired by ratiometric imaging, which can eliminate Ca(2+)-independent signal drift caused by cell shape changes, focus shift, etc. Conclusion/Significance: The high brightness and large dynamic range of BRAC should help to achieve highly sensitive Ca(2+) imaging, which is applicable not only to single live cells but also to small live animals. [4]

C26H22CLN3O2

443.92

443.1400

50909-86-9

Typically exists as Light yellow to orange solid at room temperature

4.6

3

4

5

32

555

0

C1=CC=C(C=C1)CC2=C(N3C=C(N=C(C3=N2)CC4=CC=CC=C4)C5=CC=C(C=C5)O)O.Cl

WNNCQCAQZZMPOX-UHFFFAOYSA-N

InChI=1S/C26H21N3O2.ClH/c30-21-13-11-20(12-14-21)24-17-29-25(22(27-24)15-18-7-3-1-4-8-18)28-23(26(29)31)16-19-9-5-2-6-10-19;/h1-14,17,30-31H,15-16H2;1H

2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3-ol;hydrochloride

Coelenterazine h (hydrochloride); Coelenterazine h hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~225.27 mM; with sonication)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)