Luminescent profiles of CTZ and its analogues with HSA.
This chapter describes the design of an imidazopyrazinone-type luciferin named as HuLumino1 by us and investigation of its luminescence properties. This luciferin was designed to generate bioluminescence by human serum albumin (HSA) rather than by luciferase derived from luminous organisms. HuLumino1 was developed by modifying a methoxy-terminated alkyl chain to the C-6 position and eliminating a benzyl group at the C-8 position of coelenterazine. To clarify the basis of light emission by HSA, the detailed kinetic properties of the HuLumino1/HSA pair were investigated using a calibrated luminometer. The enzymatic oxidation of HuLumino1 was observed only in the presence of HSA. Results of HSA quantification experiments using HuLumino1 agreed with less than 5% differences with those of enzyme-linked immunosorbent assays, suggesting HuLumino1 could be used for quantitative analysis of HSA levels in serum samples without any pretreatments. These results demonstrate the advantages of the coelenterazine analogue as a bioluminescence reagent to detect non-labeled proteins, which generally do not function as enzymes.
- Human serum albumin
- Quantum yield
- Enzyme-linked immunosorbent assay
Luminous organisms, such as
However, in some cases, bioluminescent or chemiluminescent substrates may induce enzymatic luminescence activity of non-bioluminescence enzymes. For example, CycLuc2, a synthetic analog of firefly luciferin, can be catalyzed to emit light by long-chain fatty acid acyl-CoA synthetase found in non-luminous insects [5, 6, 7]. In addition, the heme-containing enzyme myeloperoxidase, which is abundantly expressed in neutrophils and monocytes, can catalyze the luminescence reaction of xenobiotic luminol [8, 9]. These reports suggested that the introduction of appropriate exogenous luminescent substrates reveals luminous activity of non-bioluminescence enzymes, which can significantly differ from the conventional function of the enzyme and has potential for use in quantitative analysis of enzymes without any labeling procedures, including transgene introduction of luciferase from luminous organisms. Here, we describe the design and bioluminescence characterization of a luciferin analogue which was selectively catalyzed to exhibit bioluminescence by human serum albumin (HSA) . Serum albumins perform various physiological functions; they maintain colloid osmotic blood pressure and transport several exogenous and endogenous molecules. However, they are not categorized in the list of EC number, indicating they are not considered typical enzymes. The bioluminescence system of HSA with the luciferin analogue synthesized by us is novel and different from the conventional luciferin-luciferase reaction systems.
2. Design of coelenterazine analogue
2.1 Coelenterazine analogue with HSA-specific bioluminescence
Most luciferases from luminous marine organisms use coelenterazine (CTZ) as their luciferin to form coelenteramide in an excited state, with emission ranging from blue to green at approximately 400–500 nm (Figure 1) [11, 12]. CTZ is oxidized by bovine serum albumin (BSA) in addition to luciferase, and this has been considered as nonspecific reaction mainly occurs because of a simple luminescence reaction that requires only an oxygen molecule  (Figure 1).
The emission ability of CTZ is derived from the imidazopyrazinone ring, and the chemical structure of sidechains at the C-2, C-6, and C-8 position of the imidazopyrazinone core significantly affect enzyme recognition. For example,
To clarify the potential enzymatic luminescence activity of human proteins, we focused on HSA, which accounts for approximately 65% of serum proteins in the human body . This abundant protein is involved in a wide variety of physiological functions, such as maintaining osmotic pressure, buffering blood pH levels, and carrying ligands including hormones, amino acids, and fatty acids [17, 18]. In addition, HSA and some ligand complexes often possess enzymatic activities, such as Kemp elimination and hydrolysis of esters because of their unique ability to bind small hydrophobic molecules in some cavities; however, the potential enzymatic activities remain unclear [17, 19].
First, to obtain a rational luciferin with an imidazopyrazinone core for HSA-specific BL, we assayed CTZ and previously reported 18 CTZAs named as Bottle Blue (BBlue), where the
Based on these results, we predicted that elimination of the benzyl group at the C-8 position of BBlue2.3 would relieve its steric hindrance with key amino acids in the substrate binding site of HSA and enhance the enzymatic luminescence reaction of HSA. We then designed and synthesized a novel CTZA, named as Human Luminophore
These results suggest “luciferase” activity of HSA catalyzes the enzymatic luminescence reaction of CTZAs to produce “bioluminescence”.
2.2 Quantitative evaluation of luminescence intensity
To characterize the enzymatic luminescence reaction with HSA, the bioluminescence intensity of CTZAs and HSA pairs was quantitatively evaluated. Bioluminescence intensity is generally determined by several reaction factors including the bioluminescence quantum yield of luciferin, turnover number (
The Michaelis–Menten constant () of luciferin was calculated from Lineweaver-Burk plots constructed using a standard method. The catalytic constant (), which is the turnover number of the reaction for luciferin by a single luciferase molecule per second, was calculated from the value and maximum velocity () determined from the Lineweaver-Burk plots.
|CTZ/HSA||0.32 ± 0.03||25.3 ± 5.2||2.75 ± 0.3|
|HuLumino||30.9 ± 3.1||4.28 ± 1.24||0.30 ± 0.06|
|CTZA-4/HSA||42.2 ± 9.1||2.46 ± 0.40||0.11 ± 0.09|
2.3 Enzymatic reaction site of HSA
The crystal structure of HSA, with binding to a variety of drugs, clarified the two principal drug binding sites in different subdomains (site 1 in subdomain IIA and site 2 in subdomain IIIA) [26, 27]. To investigate the luminescent reaction site between HSA and HuLumino
Next, to investigate the effect of the steric structure of HSA on luminescence, HSA pretreated with 10 M guanidine hydrochloride, a reagent commonly used to induce denaturation of the α-helix structure of proteins , was prepared, and the luminescence of HuLumino
2.4 Bioluminescent assay for HSA
Low levels of HSA in the serum (<35 mg/mL) are biomarkers of several diseases such as malnutrition, cirrhosis, and chronic hepatitis . In hospitals, HSA is evaluated using the colorimetric bromocresol green assay or ELISA. Both can provide a reliable assessment of albumin but require sample preparation and processing time (e.g. 3 h for ELISA) . Therefore, an assay for the simple, accurate and rapid detection of HSA in the serum should be developed for clinical diagnosis. We demonstrated that the BL assay can be used to evaluate HSA based on the enzymatic luminescence reaction of HuLumino
Finally, two HSA assays, including our developed BL-based assay and ELISA, were performed to evaluate human serum from male AB plasma. The HSA levels calculated with HuLumino
|Amount of HSA added (mg/mL)||HSA (mg/mL) determined by developed methoda||HSA (mg/mL) determined by ELISA||Recovery|
|0||39.0 ± 3.1||41.0 ± 3.6||95.2|
|1||44.5 ± 0.5||ND||106.1|
|2.5||45.2 ± 0.5||ND||104.1|
We designed and synthesized the first luciferin (HuLumino
We acknowledge funding from JST, PRESTO Grant Number JPMJPR20EB, JSPS KAKENHI Grant Number 20 K15421, AMED under Grant Number JP191m0203012, Shimazu Science Foundation, Research Foundation for Opto-Science and Technology, and Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, and DAICENTER project grant from DBT (Govt. of India) to Renu Wadhwa and special strategic grant from AIST (Japan).
Conflict of interest
The authors declare no conflict of interest.
We thank Dr. Tsukasa Ishihara (Biomedical Research Institute, National Institute of Advanced Industrial International Science and Technology) for his help with the docking simulation experiment.
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