Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.
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http://dx.doi.org/10.1016/j.ab.2006.10.043 | DOI Listing |
Anal Chim Acta
February 2025
State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Gansu, Lanzhou, 730000, China. Electronic address:
The presence of lead ion (Pb) in groundwater poses a serious risk to human health, even at low levels. Therefore, it is essential to develop a new strategy for both selective detection and effective removal of Pb in groundwater, which has been rarely reported. Here, we developed a multi-functional chitosan-based fluorescent sensing membrane (CM-L/CG) by using a casting method for the sensitive/selective detection and removal of Pb in groundwater.
View Article and Find Full Text PDFAnal Biochem
January 2025
Pharmaceutical Analytical Chemistry Department, Faculty of pharmacy, Zagazig University, Zagazig 44519, Egypt.
This work represents different spectrophotometric techniques for concurrent quantification of Indacaterol (IND) and Mometasone furoate (MOM); co-formulated inhalation capsules to control asthma symptoms. Direct spectrophotometric (D) approach was applied for IND assay. While, absorption factor (AF), ratio difference (RD), mean centering of the ratio spectra (MC), and continuous wavelet transform (CW) techniques were utilized for MOM quantification.
View Article and Find Full Text PDFFront Biosci (Landmark Ed)
January 2025
Department of Cardiovascular Medicine, Binzhou Medical University Hospital, 256603 Binzhou, Shandong, China.
Background: Cellular vacuolization is a commonly observed phenomenon under physiological and pathological conditions. However, the mechanisms underlying vacuole formation remain largely unresolved.
Methods: LysoTracker Deep Red probes and Enhanced Green Fluorescent Protein-tagged light chain 3B (LC3B) plasmids were employed to differentiate the types of massive vacuoles.
Plants (Basel)
January 2025
Key Laboratory of Coarse Cereal Processing, Ministry of Agriculture and Rural Affairs, Sichuan Engineering and Technology Research Center of Coarse Cereal Industrialization, School of Food and Biological Engineering, Chengdu University, Chengdu 610106, China.
Djulis ( Koidz.), a member of the family plant, is noted for its vibrant appearance and significant ornamental value. However, the mechanisms underlying color variation in its spikes remain unexplored.
View Article and Find Full Text PDFPlants (Basel)
January 2025
Departamento de Química, Universidade Federal de Viçosa, Campus Universitário, Avenida Peter Henry Rolfs, s/n, Viçosa 36570-900, MG, Brazil.
Soxhlet extraction is a method recommended by the Association of Official Analytical Chemists (AOAC) to determine the lipid content in plant samples. Generally, n-hexane (toxicity grade 5) is used as the solvent (≈300 mL; ≈30 g sample) at boiling temperatures (69 °C) for long times (≤16 h) under a chilled water reflux (≈90 L/h), proportionally aggravated by the number of repetitions and samples determined. In this sense, the technique is neither safe nor sustainable for the analyst or the environment.
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