Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods.

Autor: Pavankumar AR; ApiRays AB, Karolinska Institute Science Park, Stockholm, Sweden., Zelenin S; Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden., Lundin A; BioThema AB, Handen, Stockholm, Sweden., Schulte T; Science for Life Laboratory, Department of Medicine Solna, Karolinska Institute, Solna, Stockholm, Sweden; Department of Infectious Diseases, Karolinska University Hospital, Solna, Stockholm, Sweden., Rajarathinam K; ApiRays AB, Karolinska Institute Science Park, Stockholm, Sweden., Rebellato P; BioThema AB, Handen, Stockholm, Sweden., Ardabili S; Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden., Salas J; ApiRays AB, Karolinska Institute Science Park, Stockholm, Sweden., Achour A; Science for Life Laboratory, Department of Medicine Solna, Karolinska Institute, Solna, Stockholm, Sweden; Department of Infectious Diseases, Karolinska University Hospital, Solna, Stockholm, Sweden., Russom A; Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden. Electronic address: aman.russom@scilifelab.se.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2018 Sep 26; Vol. 1025, pp. 118-123. Date of Electronic Publication: 2018 Apr 26.
DOI: 10.1016/j.aca.2018.04.054
Abstrakt: Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.
(Copyright © 2018 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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