Luminescence of Cypridina Luciferin in the Presence of Human Plasma Alpha 1-Acid Glycoprotein

The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in the presence of several proteins with drug-binding ability. Luminescence measurements showed that the mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produced light. The total value of the luminescence intensity over 60 s was over 12.6-fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP. Chlorpromazine, which binds to hAGP, showed an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of hAGP and a recombinant CLase. Furthermore, BlastP analysis showed that hAGP had partial amino acid sequence similarity to known CLases in the region including amino acid residues involved in the drug-binding ability of hAGP. These findings indicate enzymological similarity between hAGP and CLase and provide insights into both the enzymological understanding of CLase and development of a luminescence detection method for hAGP

Bioluminescence of (<i>R</i>)-Cypridina Luciferin with <i>Cypridina</i> Luciferase

Cypridina luciferin (CypL) is a marine natural product that functions as the luminous substrate for the enzyme Cypridina luciferase (CypLase). CypL has two enantiomers, (R)- and (S)-CypL, due to its one chiral center at the sec-butyl moiety. Previous studies reported that (S)-CypL or racemic CypL with CypLase produced light, but the luminescence of (R)-CypL with CypLase has not been investigated. Here, we examined the luminescence of (R)-CypL, which had undergone chiral separation from the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that (R)-CypL with CypLase produced light, indicating that (R)-CypL must be considered as the luminous substrate for CypLase, as in the case of (S)-CypL, rather than a competitive inhibitor for CypLase. Additionally, we found that the maximum luminescence intensity from the reaction of (R)-CypL with CypLase was approximately 10 fold lower than that of (S)-CypL with CypLase, but our kinetic analysis of CypLase showed that the Km value of CypLase for (R)-CypL was approximately 3 fold lower than that for (S)-CypL. Furthermore, the chiral high-performance liquid chromatography (HPLC) analysis of the reaction mixture of racemic CypL with CypLase showed that (R)-CypL was consumed more slowly than (S)-CypL. These results indicate that the turnover rate of CypLase for (R)-CypL was lower than that for (S)-CypL, which caused the less efficient luminescence of (R)-CypL with CypLase

Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting L-Cys[1-¹³C] or L-Cys[3-¹³C] with 1,4-hydroquinone into the adult of <i>L. lateralis.</i>

<p>^a (<b><i>a</i></b>) represents the parent mass of firefly luciferin with MH⁺281 (+0, 100%). (<b><i>b</i></b>) and (<b><i>c</i></b>) represent the fragment ion mass from firefly luciferin with MH⁺235 (+0, 100%) and MH⁺177 (+0, 100%), respectively, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084023#pone-0084023-g002" target="_blank">Fig. 2</a>. The numbers in bold are the significant mass peaks containing the incorporated stable isotope atoms.</p

Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of L-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

<div><p>Background</p><p>Bioluminescence in fireflies and click beetles is produced by a luciferase-luciferin reaction. The luminescence property and protein structure of firefly luciferase have been investigated, and its cDNA has been used for various assay systems. The chemical structure of firefly luciferin was identified as the D-form in 1963 and studies on the biosynthesis of firefly luciferin began early in the 1970’s. Incorporation experiments using ¹⁴C-labeled compounds were performed, and cysteine and benzoquinone/hydroquinone were proposed to be biosynthetic component for firefly luciferin. However, there have been no clear conclusions regarding the biosynthetic components of firefly luciferin over 30 years.</p><p>Methodology/Principal Findings</p><p>Incorporation studies were performed by injecting stable isotope-labeled compounds, including L-[U-¹³C₃]-cysteine, L-[1-¹³C]-cysteine, L-[3-¹³C]-cysteine, 1,4-[D₆]-hydroquinone, and <i>p</i>-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly <i>Luciola lateralis</i>. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was identified by LC/ESI-TOF-MS. The positions of the stable isotope atoms in firefly luciferin were determined by the mass fragmentation of firefly luciferin.</p><p>Conclusions</p><p>We demonstrated for the first time that D- and L-firefly luciferins are biosynthesized in the lantern of the adult firefly from two L-cysteine molecules with <i>p</i>-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of L-cysteine.</p></div

Identification of arbutin in <i>L. lateralis</i> by HPLC analysis.

<p>A. HPLC analysis of the extracts from an adult <i>L. lateralis</i> by using a fluorescence detector. (a) authentic arbutin, (b) the extracts of <i>L. lateralis</i> lantern. The arbutin fraction between the vertical dashed lines is used for hydrolysis as in Fig. 10C. B. HPLC analysis of the hydrolyzed arbutin fraction in Fig. 10A–b. (a) authentic 1,4-hydroquinone (labeled peak 1) containing benzoquinone (labeled peak 2), (b) the hydrolyzed products of the peak fraction between the dashed lines in Fig. 10A–b. Asterisk indicates 1,4-hydroquinone from arbutin. C. A scheme of acid hydrolysis of arbutin to 1,4-hydroquinone by acid treatment with HCl.</p

Proposed biosynthetic pathway of firefly luciferin in the lantern of adult firefly.

<p>Proposed biosynthetic pathway of firefly luciferin in the lantern of adult firefly.</p

Incorporation of 1,4-hydroquinone and ¹³C-labeled L-cysteines into firefly luciferin in an adult lantern of <i>L. lateralis</i>.

<p>A. Predicted firefly luciferins incorporated from 1,4-hydroquinone and L-Cys[U-¹³C₃]. B. Predicted firefly luciferins incorporated from 1,4-hydroquinone and L-Cys[1-¹³C₃]. C. Predicted firefly luciferins incorporated from 1,4-hydroquinone and L-Cys[3-¹³C₃]. The number in parenthesis on the right indicates the number of ¹³C-atom incorporated into firefly luciferin. Asterisk indicates the position of a ¹³C-atom.</p

Strategy to study on the biosynthetic pathway of firefly luciferin in an adult lantern of a living firefly by injecting the stable isotope-labeled compounds, and the bioluminescence reaction catalyzed by firefly/beetle luciferase.

<p>A. Proposed biosynthetic pathway of firefly luciferin (<b>I</b>) from <i>p</i>-benzoquinone and two L-cysteines in an adult lantern, and the luminescence reaction of luciferin with firefly luciferase, followed by the formation of 2-cyano-6-hydroxybenzothiazole (<b>III</b>) from oxyluciferin (<b>II</b>). B. Stable isotope-labeled L-cysteines used in the experiments. Asterisk indicates the position of a ¹³C atom. C. Preparation of <i>p</i>-[D₄]-benzoquinone from 1,4-[D₆]-hydroquinone by the oxidation reaction using silver oxide with H₂O₂.</p

Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting L-Cys[U-¹³C₃] with 1,4-hydroquinone or <i>p</i>-benzoquinone into the adult of <i>L. lateralis.</i>

<p>^a (<b><i>a</i></b>) represents the parent mass of firefly luciferin with MH⁺281 (+0, 100%). (<b><i>b</i></b>) and (<b><i>c</i></b>) represent the fragment ion mass from firefly luciferin with MH⁺235 (+0, 100%) and MH⁺177 (+0, 100%), respectively, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084023#pone-0084023-g002" target="_blank">Fig. 2</a>. The numbers in bold are the significant mass peaks containing the incorporated stable isotope atoms.</p

Incorporation of <i>p</i>-[D₄]-benzoquinone or 1,4-[D₆]-hydroquinone with L-cysteines into firefly luciferin in an adult lantern of <i>L. lateralis</i>.

<p>A. Predicted firefly luciferins incorporated from <i>p</i>-[D₄]-benzoquinone and L-cysteine. B. Predicted firefly luciferins incorporated from 1,4-[D₆]-hydroquinone and L-cysteine. C. Predicted firefly luciferins incorporated from 1,4-[D₆]-hydroquinone and L-Cys[3-¹³C₃]. The number in parenthesis on the right indicates the number of the stable isotope atoms incorporated into firefly luciferin. Asterisk indicates the position of a ¹³C-atom.</p