Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase
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Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase. / Holland, Ryan J; Paulisch, Rika; Cao, Zhao; Keefer, Larry K; Saavedra, Joseph E; Donzelli, Sonia.
in: NITRIC OXIDE-BIOL CH, Jahrgang 35, 30.11.2013, S. 131-6.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase
AU - Holland, Ryan J
AU - Paulisch, Rika
AU - Cao, Zhao
AU - Keefer, Larry K
AU - Saavedra, Joseph E
AU - Donzelli, Sonia
N1 - Copyright © 2013. Published by Elsevier Inc.
PY - 2013/11/30
Y1 - 2013/11/30
N2 - We introduce a strategy for generating mixtures of nitric oxide (NO) and nitroxyl (HNO) at tunable rates in physiological media. The approach involves converting a spontaneously HNO/NO-generating ion to a caged (prodrug) form that is essentially stable in neutral media, but that can be activated for HNO/NO release by adding an enzyme capable of efficiently opening the cage to regenerate the ion. By judiciously choosing the enzyme, substrate, and reaction conditions, unwanted scavenging of the HNO and NO by the protein can be minimised and the catalytic efficiency of the enzyme can be maintained. We illustrate this approach with a proof-of-concept study wherein the prodrug is Gal-IPA/NO, a diazeniumdiolate of structure iPrHN-N(O)NOR, with R=β-d-galactosyl. Escherichia coli-derived β-d-galactosidase at concentrations of 1.9-15nM hydrolysed 56μM substrate with half-lives of 140-19min, respectively, producing the IPA/NO anion (iPrHN-N(O)NO(-), half-life ∼3min), which in turn spontaneously hydrolysed to mixtures of HNO with NO. Using saturating substrate concentrations furnished IPA/NO generation rates that were directly proportional to enzyme concentration. Consistent with these data, the enzyme/substrate combination applied to ventricular myocytes isolated from wild-type mouse hearts resulted not only in a significant positive inotropic effect, but also rescued the cells from the negative inotropy, hypercontractions, and occasional cell death seen with the enzyme alone. This mechanism represents an alternate approach for achieving controlled fluxes of NO/HNO to investigate their biological actions.
AB - We introduce a strategy for generating mixtures of nitric oxide (NO) and nitroxyl (HNO) at tunable rates in physiological media. The approach involves converting a spontaneously HNO/NO-generating ion to a caged (prodrug) form that is essentially stable in neutral media, but that can be activated for HNO/NO release by adding an enzyme capable of efficiently opening the cage to regenerate the ion. By judiciously choosing the enzyme, substrate, and reaction conditions, unwanted scavenging of the HNO and NO by the protein can be minimised and the catalytic efficiency of the enzyme can be maintained. We illustrate this approach with a proof-of-concept study wherein the prodrug is Gal-IPA/NO, a diazeniumdiolate of structure iPrHN-N(O)NOR, with R=β-d-galactosyl. Escherichia coli-derived β-d-galactosidase at concentrations of 1.9-15nM hydrolysed 56μM substrate with half-lives of 140-19min, respectively, producing the IPA/NO anion (iPrHN-N(O)NO(-), half-life ∼3min), which in turn spontaneously hydrolysed to mixtures of HNO with NO. Using saturating substrate concentrations furnished IPA/NO generation rates that were directly proportional to enzyme concentration. Consistent with these data, the enzyme/substrate combination applied to ventricular myocytes isolated from wild-type mouse hearts resulted not only in a significant positive inotropic effect, but also rescued the cells from the negative inotropy, hypercontractions, and occasional cell death seen with the enzyme alone. This mechanism represents an alternate approach for achieving controlled fluxes of NO/HNO to investigate their biological actions.
U2 - 10.1016/j.niox.2013.10.003
DO - 10.1016/j.niox.2013.10.003
M3 - SCORING: Journal article
C2 - 24126017
VL - 35
SP - 131
EP - 136
JO - NITRIC OXIDE-BIOL CH
JF - NITRIC OXIDE-BIOL CH
SN - 1089-8603
ER -