Pholasin--a bioluminescent indicator for detecting activation of single neutrophils |
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| Authors: | P A Roberts J Knight A K Campbell |
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| Institution: | 1. Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, United Kingdom;1. The Laboratory, Western College Road, Plymouth, PL4 7AG, United Kingdom;1. School of Basic Sciences, Indian Institute of Technology Mandi, Kamand Campus, Mandi, HP 175005, India;2. Institute of Biophysics and Centre of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover, Germany;3. Institute of Technical Chemistry and Centre of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Callinstrasse 5, 30167 Hannover, Germany;4. Institute of Organic Chemistry and Centre of Biomolecular Drug Research (BMWZ), Leibniz Universität Hannover, Schneiderberg 1B, 30167 Hannover, Germany;5. School of Chemical Sciences, National Institute of Science Education and Research Bhubaneswar, Jatni Campus, Kurdha, Odisha, 752050, India;1. Department of Chemistry, Faculty of Science, The University of Maroua, P. O. Box 46, Maroua, Cameroon;2. H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan;3. Department of Chemistry, Higher Teachers’ Training College, University of Maroua, P. O. Box 55, Maroua, Cameroon;4. Medical Research and Applied Biochemistry Laboratory, University of Buea, P. O. Box 63, Buea, Cameroon;5. Faculty of Sciences, Department of Chemistry, University of Douala, 24157, Douala, Cameroon;6. Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany;7. Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi Arabia;1. Key Laboratory of TCM Quality Control Technology of Shandong Province, Shandong Analysis and Test Center, 19 Keyuan Street, Jinan, Shandong 250014, PR China;2. Reyoung Pharmaceutical Co., Ltd., No. 44Cultural West Road, Jinan 250012, PR China;1. Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt;2. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mu’tah University, 61710 Al-Karak, Jordan;3. Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt |
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| Abstract: | Pholasin is the protein-bound luciferin from the bivalve mollusc Pholas dactylus which reacts with its luciferase and molecular oxygen to produce light. Pholasin was purified 226-fold with a yield of 58% from P. dactylus to give a preparation free from luciferase contamination. The ratio (k) of endogenous pholasin chemiluminescence to that when maximally stimulated by luciferase was 8.12 X 10(-6) +/- 0.87 X 10(-6) (mean +/- SD, n = 6), equivalent to a t 1/2 of 23.7 h at pH 9. Pholasin could detect reactive oxygen metabolite production from neutrophils stimulated by the chemotactic peptide N-formyl-Met-Leu-Phe, in the presence and absence of 2-chloroadenosine or cytochalasin B, and by latex beads in the presence and absence of cytochalasin B. Pholasin was also able to detect a longer-lived oxidative activity distinct from myeloperoxidase, and released from neutrophils activated by latex beads or chemotactic peptide; luminol could not. Under optimal conditions pholasin produced a signal some 50-100 times that of luminol in the presence of activated neutrophils. This enabled activation of a single neutrophil by chemotactic peptide (1 microM) to be detected, giving a signal of 194 +/- 21 chemiluminescent counts per second, some six times that of the background signal (mean +/- SD, n = 2). Pholasin thus provides an indicator sufficiently sensitive to establish whether neutrophil activation occurs through thresholds in individual cells. |
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