Laccase–luminol chemiluminescence system: an investigation of substrate inhibition

• Published in: Luminescence (Chichester, England) Vol. 38; no. 3; pp. 341 - 349
• Main Authors: Sánchez‐Trasviña, Calef, Galindo‐Estrada, José Daniel, Tinoco‐Valencia, Raunel, Serrano‐Carreón, Leobardo, Rito‐Palomares, Marco, Willson, Richard C., Mayolo‐Deloisa, Karla
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.03.2023
• Subjects: biosensor; Chemiluminescence; Detection; Emission analysis; Enzyme kinetics; Kinetics; Laccase; Laccase - chemistry; Light emission; Luminescence; Luminescent Measurements; luminol; Luminol - chemistry; Sodium hydroxide; Substrate inhibition; Substrates; biosensor; substrate inhibition; chemiluminescence; laccase; luminol
• ISSN: 1522-7235; 1522-7243
• DOI: 10.1002/bio.4460

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase–luminol system, determine its kinetic parameters, and analyze the effects of protein and OHˉ concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase–luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate‐inhibition behaviour. A two‐site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase–luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification. Laccase–luminol system can produce light emission without intermediate molecules. The laccase–luminol system exhibited substrate‐inhibition behaviour with maximum light intensity at 50 μM of luminol. The kinetic modelling suggested that once the first luminol molecule is bound to the active site; the laccase affinity for the second luminol molecule increases, therefore enhancing the substrate inhibitory effect.