Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase

Article abstract of DOI:10.1016/j.niox.2013.10.003

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 1.9-15 nM hydrolysed 56 μM substrate with half-lives of 140-19 min, respectively, producing the IPA/NO anion (iPrHN-N(O)NO^-, half-life ～3 min), 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.

Full text of DOI:10.1016/j.niox.2013.10.003

Nitric Oxide 35 (2013) 131–136
Contents lists available at ScienceDirect
Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox
Enzymatic generation of the NO/HNO-releasing IPA/NO anion
at controlled rates in physiological media using b-galactosidase
Ryan J. Holland a, ,1, Rika Paulisch b,1, Zhao Cao c, Larry K. Keefer a, Joseph E. Saavedra c, Sonia Donzelli b
⇑
a Drug Design Section, Chemical Biology Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA
b Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
c Basic Research Program, SAIC-Frederick, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 struc-
Received 3 August 2013
Revised 19 September 2013
Available online 11 October 2013
Keywords:
IPA/NO
Prodrug
Nitroxyl
Diazeniumdiolate
Gal-IPA/NO
b-Galactosidase
ture iPrHN–N(O)@NOR, with R = b-
tions of 1.9–15 nM hydrolysed 56
D
-galactosyl. Escherichia coli-derived b-
D-galactosidase at concentra-
lM substrate with half-lives of 140–19 min, respectively, producing
the IPA/NO anion (iPrHN–N(O)@NOꢀ, half-life ꢁ3 min), which in turn spontaneously hydrolysed to mix-
tures 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, hypercon-
tractions, 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.
Ó 2013 Published by Elsevier Inc.
Introduction
meaning that it cannot be isolated in pure form. This problem
can be circumvented by employing prodrug forms such as Angeli’s
A
variety of research tools are available for generating
salt (AS) [8,9], a well-established HNO prodrug that in our hands
hydrolyses to a 93:7 mixture of HNO to NO in physiological buffer
with a reliable half-life of ꢁ3 min at physiological temperature and
pH.
controlled fluxes of the multifaceted bioregulatory agent nitric
oxide (NO) in physiological media, including a series of ionic and
zwitterionic diazeniumdiolates whose reproducible half-lives for
NO release range from 2 s to 20 h at pH 7.4 and 37 °C, depending
on their structure. These agents have greatly aided the effort to
characterise NO’s biological activities over the last 20 years [1,2].
More recently, nitroxyl (HNO, the product of reducing NO by
one electron) has been shown to possess intriguing biological
properties, some of potential therapeutic significance [3–7].
However, there are several properties of HNO that have compli-
cated attempts to study its chemistry and pharmacology. One
problem is that it rapidly dimerises and self-destructs Eq. (1),
2HNO ! N2O þ H2O
ð1Þ
AS has provided many major insights into the chemical biology
of HNO [9–13], but it has not been ideal for studying the effects of
HNO fluxes much longer than the 3-min half-life of AS can provide.
Recently, several alternate HNO prodrugs have been introduced
that spontaneously hydrolyse to produce HNO in high yield with
discrete half-lives of seconds to hours, depending on their struc-
ture [14–19]. These produgs have provided an increasingly valu-
able set of research tools as their respective by-product reactivity
and toxicity profiles are elucidated.
Here we describe a system that delivers both HNO and NO
simultaneously over an infinitely tunable range of half-lives. It
involves the use of stable prodrugs that in presence of an activating
enzyme undergo two-step hydrolysis releasing both potent
Abbreviations: NO, nitric oxide; IPA/NO, sodium 1-(isopropylamino)diazen-1-
ium-1,2-diolate; HNO, nitroxyl.
⇑
Corresponding author. Fax: +1 301 846 5946.
E-mail address: hollandrj@mail.nih.gov (R.J. Holland).
These authors contributed equally to this work.
1
1089-8603/$ - see front matter Ó 2013 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.niox.2013.10.003

132
R.J. Holland et al. / Nitric Oxide 35 (2013) 131–136
bioregulators, and any downstream cross talk products, at rates
that can be precisely programmed.
b-galactosidase at 37 °C was sparged with inert gas until a steady
detector response was established. IPA/NO or Gal-IPA/NO were
added to a final concentration of 56 lM and the NO release profile
was followed over time after injection. The resulting curve was
integrated to quantify the amount of NO released/mol of
compound.
Materials and methods
Chemicals
Griess assay test for nitrite detection
Liberase TM (Thermolysin medium) Research Grade Enzyme
was obtained from Roche Diagnostics (Mannheim). Fetal calf
serum (FCS) was purchased from Invitrogen (Molecular Probes™,
Karlsruhe). All other chemicals used in the experiment (including
b-galactosidase from Escherichia coli) were acquired from Sigma
to Aldrich (St. Louis, MO, USA), except CaCl2 (Merck, Darmstadt).
Substrate/enzyme reactions were allowed to proceed to com-
pletion in the absence of purging. We then added 100
lL of Griess
reagent, 300 L of sample and 2.6 mL of deionised water together
l
in a spectrophotometer cuvette. We incubated the mixture for
30 min at room temperature and prepared a reference sample by
mixing 100 lL of Griess reagent and 2.9 mL of deionised water.
O2-(2,3,4,6-tetra-O-acetyl-b-
D-galactopyranosyl) 1-(N-
isopropylamino)diazen-1-ium-1,2-diolate
The absorbance of the nitrite-containing sample at 548 nm relative
to the reference sample was converted to nitrite concentrations
using a calibration curve.
A solution of 2.64 g (0.0064 mol) of acetobromo-a-D-galactose
in 100 mL of acetone was cooled to 0 °C. A solution of 1.06 g
(0.0075 mol) of IPA/NO in 50 mL of cold (5 °C) 5% aqueous sodium
bicarbonate was placed in a dropping funnel equipped with an ice-
jacket and added dropwise to the acetone solution. The reaction
mixture was allowed to warm up to room temperature gradually
and stirred for 48 h. The acetone was then removed on a rotary
evaporator and the remaining aqueous portion was extracted with
dichloromethane. The organic layer was separated, dried over
sodium sulfate, filtered through magnesium sulfate and concen-
trated under vacuum to give 1.22 g of a syrup. The crude product
was chromatographed on an 80-g silica gel pre-packed column
and eluted with dichloromethane:ethyl acetate to give 370 mg
N2O measurements by gas chromatography
Reactions were run according to the conditions stated above.
The gas chromatography was performed on a Shimadzu GC-2014
with an electron capture detector, equipped with 63Ni 370 MBq
source. A Restek ShinCarbon 80/100 packed column (2 m ꢂ 2.0 mm
ID) was used with helium as carrier gas. The GC operation condi-
tions were as follows: injector and detector temperatures were at
250 °C, oven temperature was programmed from 90 to 200 °C at
20 °C/min and held at 200 °C for 1.1 min. Helium flow was
30 mL/min and nitrogen was used as makeup gas at 2 mL/min.
(13%) of O2-(2,3,4,6-tetra-O-acetyl-b-
propylamino)diazen-1-ium-1,2-diolate as
several preparations following this procedure produced a total of
) 238 nm
D-galactopyranosyl) 1-(N-iso-
a
crystalline solid;
Kinetic studies
1.39 g of material: mp 36–8 °C; UV (ethanol) kmax (e
Kinetic experiments were performed at 37 °C using a standard
UV–visible spectrophotometer. Reactions were initiated by addi-
tion of substrate after the buffer and enzyme reached thermal
(6.9 mMꢀ1 cmꢀ1); 1H NMR (CDCl3) d 1.19 (d, 6H) J = 6.25 Hz, 1.99
(s, 3H), 2.05 (s, 3H), 2.05 (s, 6H), 2.16 (s, 3H), 3.97–4.01 (m, 1H),
4.11–4.18 (m, 2H), 5.07–5.14 (m, 2H), 5.42–5.43 (m, 1H), 5.48–
5.52 (m, 1H), 6.16 (d, 1H) J = 8.99 Hz; 13C NMR (CDCl3) d 20.40,
20.42, 20.49, 20.58, 20.61, 49.20, 61.06, 66.63, 66.78, 70.91,
71.44, 100.84, 169.02, 170.02, 170.11, 170.31. HRMS (ESI) m/z cal-
equilibrium. Typical substrate concentrations were 56
b-galactosidase concentration range of 2–15 nM in 0.1 M phos-
phate buffer, pH 7.4, containing 50 M diethylenetriamine pentaa-
lM with a
l
cetic acid (DTPA). In each experiment the data were analysed at
247 nm and the rate was derived by fitting the data to an exponen-
tial curve typical for first order processes.
culated for
C
17H27N3O11 (M+H)+ 450.1784, found 450.17131,
D
ppm = 1.04.
O2-b-
D-Galactopyranosyl) 1-(N-isopropylamino)diazen-1-ium-1,2-
Analysis for amine and alcohol products
diolate (Gal-IPA/NO)
b-Galactosidase was stirred in 1 mL of 0.1 M phosphate buffer,
To a solution of 712 mg (1.59 mmol) of the above tetraacetate in
75 mL of methanol was added 80 lL of 25% sodium methoxide in
pH 7.4, containing 50
(DTPA), at 37 °C in a sealed cuvette. To this solution was added
12 mg of Gal-IPA/NO for a final concentration of 6.6 M. The
decomposition of Gal-IPA/NO was followed by UV/vis spectros-
copy. Upon completion the solution was cooled to 4 °C prior to
opening the cuvette. It was then filtered through a YM-3 microcon
centrifugation filter and added to an NMR tube with 10% D2O.
Spectra were run on a Varian Inova 400 MHz NMR with a Dell Pre-
cision 390 workstation. The samples were run at 37 °C and water
suppression was achieved by using the preset pulse sequence.
lM diethylenetriamine pentaacetic acid
methanol; the mixture was stirred for 2 h at room temperature.
To the solution was added 2 g of washed Amberlist 15-H+; the mix-
ture was swirled for a few minutes, filtered, and evaporated under
vacuum to give 568 mg of product as a white glassy substance that
became crystalline on storage at ꢀ10 °C: mp 54–56 °C; UV (PBS)
l
kmax (e
) 237 nm (7.2 mMꢀ1 cmꢀ1); 1H NMR (CD3OD) d 1.05 (d,
6H) J = 6.3 Hz, 3.45–3.47 (m, 2H), 3.67–3.70 (m, 3H), 3.78–3.83
(m, 3H), 4.81 (d, 1H) J = 9.2 Hz; 13C NMR (CD3OD) d 18.74, 60.83,
68.56, 69.04, 73.38, 75.89, 103.86. HRMS (ESI) m/z calculated for
C9H20N3O7 (M+H)+ 182.12958, found 182.12881,
Dppm = 4.2.
Animals
Analysis for NO
Mice (3–4 months of age) from a black Swiss background were
used in this study. Animal care and experimental procedures were
performed in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH Pub. No. 85-23, revised 1996) and
were approved by the governmental review board in Hamburg
(G21/1-46/04).
Chemiluminescence detection and quantification of NO evolv-
ing from the reactions were conducted using a Sievers 280i nitric
oxide analyser (NOA). A pH 7.4 solution of 0.1 M phosphate buffer
with 50 lM diethylenetriamine pentaacetic acid (DTPA) containing

R.J. Holland et al. / Nitric Oxide 35 (2013) 131–136
133
Sarcomere shortening and Ca2+ transients measurements in isolated
ventricular myocytes
to that of Angeli’s salt [21,22]. Although hydrolysis of IPA/NO at
physiological pH yields a lower HNO/NO ratio among the products
(Fig. 1), the two anions nevertheless compare and contrast
favorably with each other in some important ways. Of special
importance in the present connection, IPA/NO can be easily O-der-
ivatised by reaction with a diversity of protecting groups [23], each
of which can be envisioned to cleave under activating conditions to
free the IPA/NO anion in situ.
Ventricular myocytes were isolated as previously described
[20]. The isolated myocytes were preincubated in IonOptix solu-
tion (in mmol/L: 135 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2
MgSO4, 1.25 CaCl2, 20 glucose, 10 hepes, pH 7.46), containing
1 l
mol/L Fura-2-AM for 30 min. Sarcomere shortening and Ca2+
transients were simultaneously assessed on field stimulation
(1 Hz with 4-ms duration, 15 V) using a video-based sarcomere
length detection system (IonOptix Corporation) maintained at
37 °C by a heater (IonOptix) attached under the chamber.
After preliminary consideration of numerous prodrug candi-
dates, we chose Gal-IPA/NO as substrate to investigate HNO release
from an enzymatic reaction. Gal-IPA/NO was synthesized by react-
ing IPA/NO sodium salt with tetraacetylated galactosyl bromide.
This intermediate product was exposed to methanolic sodium
methoxide to remove the acetyl groups, as summarised in
Fig. 2A. The synthesized Gal-IPA/NO hydrolysed only slowly in
0.1 M phosphate at pH 7.4 and 37 °C, with a half-life of greater than
two weeks.
Statistical analysis
Paired Student t-test was used to compare sarcomere length
shortening before and after treatment. Results are presented as
mean SEM, with n indicating the number of myocytes for each
experiment. Differences between sarcomere shortening before
and after treatment were considered significant when p < 0.05.
Analysis of the data and plotting of the figures were done using
Graphpad Prism 5.0 software.
Hydrolysis of Gal-IPA/NO is first order in enzyme under saturating
conditions
We found that b-D-galactosidase isolated from E. coli very effi-
ciently cleaved Gal-IPA/NO at pH 7.4 and 37 °C (Fig. 2B). The reac-
tion, as followed by HPLC, was cleanly first order in enzyme at a
Results
saturating substrate concentration of 56
1.9–15 M range (see Fig. 3).
lM with enzyme in the
l
Prodrug synthesis
Enzyme integrity and catalytic efficiency were not affected by
exposure to Gal-IPA/NO and its hydrolysis products
Theoretically, the half-life of AS could be extended by modifying
its structure as shown in Eq. (2) to produce derivatives whose R
groups can be programmed for cleavage under controlled condi-
tions to regenerate the AS anion at the desired rate. Unfortunately,
attempts to prepare such protected derivatives of Angeli’s salt have
to our knowledge been uniformly disappointing. Even if AS could
be doubly O-derivatised to form RO–N(O) = NOR as shown, it
would presumably need two different R–O bond-breaking steps
to revert to the desired dianion, and the intermediate half-deriva-
tised species may fragment spontaneously via some unpredicted
pathway leading to products other than Angeli’s anion.
We checked for possible suicide inhibition of the galactosidase
in these experiments. No evidence for this unwanted effect was
found. The hydrolysis of 56 lM Gal-IPA/NO by 15 lM enzyme
was cleanly first order through five half-lives, with no evidence
of impaired activity at the later time points (Fig. 4, left-most trace).
The reaction was twice repeated, each time by restoring the
substrate concentration to 56 lM in the same solution in the same
cuvette, with the same observed rate constant of 1.5 ꢂ 10ꢀ3 sꢀ1
(Fig. 4) for all three reactions. There may have been a slight trend
Na2N2O3 þ 2RX ! RO—NðOÞ@N—OR þ 2NaX
ð2Þ
toward decreasing rate in run
3
(1.50 ꢂ 10ꢀ3 sꢀ1 vs.
Accordingly, we have chosen to develop this approach using
IPA/NO, a diazeniumdiolate anion that hydrolyses to a mixture of
NO and HNO in physiological buffer (Fig. 1) with a half-life similar
1.52 ꢂ 10ꢀ3 sꢀ1 for the first two), and some ill-defined background
absorbance began to accumulate over time, but we did not see a
significant drop in catalytic efficiency through the first 11,200
Fig. 1. Similarities and differences between Angeli’s salt and IPA/NO: hydrolysis rates and product profiles. Note that each of these ions hydrolyses via two mechanisms
running in parallel: HNO-producing path a and NO-generating path b.

134
R.J. Holland et al. / Nitric Oxide 35 (2013) 131–136
Fig. 2. (A) Synthesis of Gal-IPA/NO. (B) Enzymatic hydrolysis of Gal-IPA/NO with b-D-galactosidase.
Fig. 4. Effect of repeated substrate additions on catalytic efficiency. A phosphate
buffer solution (0.1 M, pH 7.4) containing 50 M diethylenetriamine pentaacetic
acid and 15 b-galactosidase was placed in cuvette held at 37 °C.
concentrated solution of Gal-IPA/NO was added with stirring to give an initial
concentration of 56 M and loss of absorbance at 247 nm was followed as a
function of time. The data were normalised to the absorbance at t0, yielding the blue
trace shown at the left. When substrate hydrolysis was effectively complete, Gal-
IPA/NO was added to the same cuvette to again give an initial concentration of
Fig. 3. The rate of IPA/NO release from 56
proportional to enzyme concentration under conditions suitable for studying the
l
M
Gal-IPA/NO proved directly
l
lM
a
A
chemical biology of HNO/NO mixtures (37 °C in 0.1 M phosphate buffer, pH 7.4,
containing 50
R
l
M diethylenetriamine pentaacetic acid). y = 4 ꢂ 10ꢀ5 ꢂ +6 ꢂ 10ꢀ6
.
l
2 = 0.9985.
56 lM, leading to the red trace. The process was repeated one more time, leading to
turnovers. Additionally, there was no detectable change in the en-
zyme’s high resolution mass spectrum after a round of catalysis.
the green curve. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
Product distribution was similar to that of IPA/NO anion hydrolysis
Effect of varying IPA/NO generation rate on myocyte contractility
Gases formed were analysed by gas chromatography for N2O as
a measure of HNO yield and, separately, conducted into a chemilu-
minescence analyser to quantify NO. Nitrite was assayed using the
Griess procedure. Organic products of Gal-IPA/NO hydrolysis were
estimated by NMR. Isopropylamine is produced through NO-yield-
ing path b of Fig. 1 (bottom reaction), while isopropanol is concur-
rently produced through the protonation, dehydration, and
hydrolysis of the diazoate ion generated via path a. The data of Ta-
ble 1 confirm the similarity of product profiles on hydrolyzing IPA/
NO vs. its galactosylated derivative.
IPA/NO has been shown to possess positive inotropism, i.e., the
ability to strengthen the beating of isolated cardiac myocytes, an
effect to be expected of prodrugs that generate HNO [21]. Here,
we report that Gal-IPA/NO is hydrolysed by b-D-galactosidase to
generate free IPA/NO anion and thence HNO in admixture with
NO. To apply this chemistry to a biological system, we chose iso-
lated cardiac myocytes, a well known and characterised target of
HNO. Isolated cardiac myocytes treated simultaneously with
Gal-IPA/NO (56
lM) and b-D-galactosidase (15 lM) at a constant
temperature of 37 °C showed initially a significant increase in

R.J. Holland et al. / Nitric Oxide 35 (2013) 131–136
135
Table 1
Products (% of theoretical) of exhaustively hydrolyzing IPA/NO vs. Gal-IPA/NO in presence or absence of b-D-galactosidase at pH 7.4 and 37 °C in the presence of 50 lM
diethylenetriamine pentaacetic acid.a
Analyte
Gal-IPA/NO + enzyme
IPA/NO, no enzyme
IPA/NO + enzyme
N2O
NO
Nitrite
Isopropylamine
Isopropanol
60 1.6
29 2.1
9.3 1.1
ꢁ30
68 1.1
30 0.3
2.7 0.3
–
64 0.5
32 0.6
3.9 0.5
–
ꢁ70
–
–
a
Values given are means standard deviations for three replicates each. ‘‘% Of theoretical’’ refers to the number of moles measured for a given analyte divided by the
number of moles that would have been seen in the absence of competing pathways (e.g., the parallel hydrolysis mechanisms represented by paths a and b in Fig. 1).
sarcomere shortening (maximum change versus baseline, from
8.3% to 11.1%, net change of 2.8%) that was completely reversed
20 min later (final condition, from 11.1% to 5.5%, Fig. 5). Next, we
inactivated half of the enzyme by incubating it at 70 °C for
10 min and treated the myocytes as above. As expected, the max-
imum increase on sarcomere shortening was only about two thirds
that seen with the fully activated enzyme (6.6% increasing to 8.3%,
or an increase of 1.7% compared to 2.8%). The increase in this case
did not reach statistical significance, though the trend was clear
(Fig. 5).
Discussion
Starting with the pioneering work of Nagasawa and colleagues,
investigators have developed various enzyme/prodrug methods for
generating nitroxyl to study its biological effects [19,24–26]. Here
we show how one such prodrug can be used for reliably tuning the
reaction rate.
Design considerations
Surprisingly, b-D-galactosidase alone induced toxicity in the
Several factors were considered in developing this strategy for
programming the rate of IPA/NO generation in solution by convert-
ing it to prodrug form and varying the concentration of a metabo-
lizing enzyme. Ideally, the caged IPA/NO donor molecule would be
totally stable in the reaction medium, fragmenting to initiate IPA/
NO generation only when the enzyme is added. The enzyme should
be unreactive with HNO, NO, and the other products of
metabolism; any reaction of HNO or NO with the protein would
artifactually diminish the amount available and potentially also
compromise the catalytic efficiency of the enzyme. Accordingly,
the catalytic efficiency of the enzyme should be as high as possible,
presenting a smaller protein target for this unwanted loss of HNO/
NO to the system. After considering a variety of enzyme/substrate
combinations, we chose b-galactosidase from E. coli with galac-
tosylated IPA/NO (Gal-IPA/NO) as substrate for these initial
proof-of-concept studies.
myocytes. In this case, a significant immediate drop in sarcomere
shortening relative to baseline was observed (from 7.4% to 3.3%),
followed by a gradual but incomplete recovery at the end of the
20-min observation period. This toxic insult was prevented by
the simultaneous treatment of the myocytes with Gal-IPA/NO
and b-D-galactosidase (Fig. 5). At first glance this immediate re-
sponse to treatment was hard to rationalise as the kinetic studies
indicate that HNO and NO should not be present in significant
quantities at such short time points. What then is responsible for
preventing the initial negative inotropism? We speculate that this
observation may be due to an ability of the cells to metabolise
Gal-IPA/NO by pathways independent of the exogenous b-D-galac-
tosidase. Support for this hypothesis was found in the Gal-IPA/NO
control (Fig. 5). When isolated cardiac myocytes were exposed to
Gal-IPA/NO alone, a slight increase in sarcomere shortening was
observed. This result is consistent with the view that Gal-IPA/NO
is, to some extent, metabolised by endogenous enzymes upon
treatment of the myocytes.
Solution chemistry
As shown in Fig. 3, the rate at which 56
hydrolysed proved linearly dependent on enzyme concentration
over the range 15–1.9 M, furnishing reproducible half-lives for
lM Gal-IPA/NO was
l
IPA/NO release of 19–140 min. Quantitative yields of HNO/NO
were estimated and found to be in ratios consistent with previous
studies of IPA/NO in our hands. There was no evidence for destruc-
tive interaction between enzyme and substrate; the rate constant
remained essentially undiminished through 11,200 turnovers and
peaks suggestive of adduct formation were absent from the mass
spectrum of the reisolated enzyme.
Proof of concept. Use of this approach in chemical biology applications
IPA/NO has been shown to exhibit positive inotropism, i.e., to
strengthen the beating of isolated cardiac myocytes, an effect to
be expected of prodrugs of HNO [21]. The present results provide
a way to extend the time course of IPA/NO’s normal 3-min half-life
for co-generating HNO and NO by converting the anion to a hydro-
lytically stable derivative, Gal-IPA/NO, that can generate free IPA/
NO anion into the medium at precisely tunable rates in a reaction
that is first order in b-D-galactosidase at saturating substrate
concentration. When this prodrug/enzyme mixture was added
simultaneously to a chamber containing isolated cardiac myocytes
Fig. 5. Effect of Gal-IPA/NO and b-D-galactosidase on myocyte shortening in
isolated cardiac myocytes. The basal condition levels were established within two
to ten minutes after myocyte selection and mounting. The final condition bars
reflect the average contractility at the 20-min time point. The middle bar provides
the point of maximum myocyte length observed under the conditions described.
The number of myocytes used for each experiment was 20, 7, 10, and 12 for each x-
axis entry, respectively. ⁄⁄⁄⁄p < 0.0001 vs. basal, ns = not significant (p > 0.05).

136
R.J. Holland et al. / Nitric Oxide 35 (2013) 131–136
[9] P. Pagliaro, D. Gattullo, C. Penna, Nitroglycerine and sodium trioxodinitrate:
from the discovery to the preconditioning effect, J. Cardiovasc. Med.
(Hagerstown) 14 (2013) 698–704.
[10] C.H. Switzer, W. Flores-Santana, D. Mancardi, S. Donzelli, D. Basudhar, L.A.
Ridnour, K.M. Miranda, J.M. Fukuto, N. Paolocci, D.A. Wink, The emergence of
undergoing electrically stimulated contraction, the intensity of
sarcomere shortening was about 50% greater when the amount
of active (non-denatured) enzyme was doubled, and the enzyme/
prodrug combination largely abrogated the cellular toxicity seen
with one of the enzyme batches we used. It is worth noting that
the positive inotropy observed in this experiment would likely
have been even more positive if the enzyme batch we used had
not been toxic.
nitroxyl (HNO) as
(2009) 835–840.
a pharmacological agent, Biochim. Biophys. Acta 1787
[11] N. Paolocci, D.A. Wink, The shy Angeli and his elusive creature: the HNO route
to vasodilation, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H1217–H1220.
[12] W. Flores-Santana, D.J. Salmon, S. Donzelli, C.H. Switzer, D. Basudhar, L.
Ridnour, R. Cheng, S.A. Glynn, N. Paolocci, J.M. Fukuto, K.M. Miranda, D.A.
Wink, The specificity of nitroxyl chemistry is unique among nitrogen oxides in
biological systems, Antioxid. Redox Signal. 14 (2011) 1659–1674.
[13] J.C. Irvine, B.K. Kemp-Harper, R.E. Widdop, Chronic administration of the HNO
donor Angeli’s salt does not lead to tolerance, cross-tolerance, or endothelial
dysfunction: comparison with GTN and DEA/NO, Antioxid. Redox Signal. 14
(2011) 1615–1624.
[14] X. Sha, T.S. Isbell, R.P. Patel, C.S. Day, S.B. King, Hydrolysis of acyloxy nitroso
compounds yields nitroxyl (HNO), J. Am. Chem. Soc. 128 (2006) 9687–9692.
[15] M.E. Shoman, J.F. DuMond, T.S. Isbell, J.H. Crawford, A. Brandon, J. Honovar,
D.A. Vitturi, C.R. White, R.P. Patel, S.B. King, Acyloxy nitroso compounds as
nitroxyl (HNO) donors: kinetics, reactions with thiols, and vasodilation
properties, J. Med. Chem. 54 (2011) 1059–1070.
We take these results as a proof-of-concept study suggesting
the utility of such approaches for co-generating NO and HNO at
controllable rates for characterizing the chemical biology of mixed
fluxes of these two bioeffector molecules. Additionally, this
approach could be used to provide insight into the effects of the
highly reactive N2Oꢀ radical anion, which is formed by direct reac-
2
tion of NO with HNO. By simultaneously generating NO and HNO at
known rates and applying the published [27,28] rate constants for
formation of N2Oꢀ and its subsequent rapid reaction with NO to
2
generate the N3Oꢀ ion, the momentary fluxes of N2Oꢀ and N3Oꢀ
3
2
3
[16] G. Keceli, J.P. Toscano, Reactivity of nitroxyl-derived sulfinamides,
Biochemistry 51 (2012) 4206–4216.
[17] D.A. Guthrie, N.Y. Kim, M.A. Siegler, C.D. Moore, J.P. Toscano, Development of
N-substituted hydroxylamines as efficient nitroxyl (HNO) donors, J. Am. Chem.
Soc. 134 (2012) 1962–1965.
should be calculable. Disproportionation of the latter species
would lead to equimolar amounts of N2O and nitrite ion, both of
which are observed in the reactions studied here (Table 1).
[18] Z. Huang, J. Kaur, A. Bhardwaj, N. Alsaleh, J.A. Reisz, J.F. DuMond, S.B. King, J.M.
Seubert, Y. Zhang, E.E. Knaus, O2-sulfonylethyl protected isopropylamine
diazen-1-ium-1,2-diolates as nitroxyl (HNO) donors: synthesis, beta-
elimination fragmentation, HNO release, positive inotropic properties, and
blood pressure lowering studies, J. Med. Chem. 55 (2012) 10262–10271.
[19] J.F. DuMond, M.W. Wright, S.B. King, Water soluble acyloxy nitroso
compounds: HNO release and reactions with heme and thiol containing
proteins, J. Inorg. Biochem. 118 (2013) 140–147.
Acknowledgments
This research was supported in part by the Intramural Research
Program of the National Institutes of Health, National Cancer Insti-
tute, Center for Cancer Research, and with Federal funds from the
National Cancer Institute under Contract HHSN261200800001E
to SAIC. We thank Dr. Sergey Tarasov and Ms. Marzena A. Dyba
of the Biophysics Resource in the Structural Biophysics Laboratory,
NCI-Frederick, for assistance with the high resolution mass spec-
trometry studies. We gratefully acknowledge Mr. John Klose for
assistance with the NMR experiments.
[20] T.D. O’Connell, Y.G. Ni, K.-M. Lin, H. Han, Z. Yan, Isolation and culture of adult
mouse cardiac myocytes for signaling studies. AfCS Research Reports Vol 1:
No.
5 CM, (2003). http://www.signaling-gateway.org/reports/v1/CM0005/
CM0005.htm.
[21] K.M. Miranda, T. Katori, C.L. Torres de Holding, L. Thomas, L.A. Ridnour, W.J.
McLendon, S.M. Cologna, A.S. Dutton, H.C. Champion, D. Mancardi, C.G.
Tocchetti, J.E. Saavedra, L.K. Keefer, K.N. Houk, J.M. Fukuto, D.A. Kass, N.
Paolocci, D.A. Wink, Comparison of the NO and HNO donating properties of
diazeniumdiolates: primary amine adducts release HNO in vivo, J. Med. Chem.
48 (2005) 8220–8228.
[22] D.J. Salmon, C.L. Torres de Holding, L. Thomas, K.V. Peterson, G.P. Goodman, J.E.
Saavedra, A. Srinivasan, K.M. Davies, L.K. Keefer, K.M. Miranda, HNO and NO
release from a primary amine-based diazeniumdiolate as a function of pH,
Inorg. Chem. 50 (2011) 3262–3270.
[23] J.E. Saavedra, D.S. Bohle, K.N. Smith, C. George, J.R. Deschamps, D. Parrish, J.
Ivanic, Y.-N. Wang, M.L. Citro, L.K. Keefer, Chemistry of the diazeniumdiolates.
O- versus N-alkylation of the RNH[N(O)NO]ꢀ ion, J. Am. Chem. Soc. 126 (2004)
12880–12887.
[24] M.J.C. Lee, H.T. Nagasawa, J.A. Elberling, E.G. DeMaster, Prodrugs of nitroxyl as
inhibitors of aldehyde dehydrogenase, J. Med. Chem. 35 (1992) 3648–3652.
[25] M.J.C. Lee, D.W. Shoeman, D.J.W. Goon, H.T. Nagasawa, N-
References
[1] R. Scatena, P. Bottoni, A. Pontoglio, B. Giardina, Pharmacological modulation of
nitric oxide release: new pharmacological perspectives, potential benefits and
risks, Curr. Med. Chem. 17 (2010) 61–73.
[2] L.K. Keefer, Fifty years of diazeniumdiolate research. From laboratory curiosity
to broad-spectrum biomedical advances, ACS Chem. Biol. 6 (2011) 1147–1155.
[3] K.M. Miranda, H.T. Nagasawa, J.P. Toscano, Donors of HNO, Curr. Top. Med.
Chem. 5 (2005) 649–664.
[4] J.C. Irvine, R.H. Ritchie, J.L. Favaloro, K.L. Andrews, R.E. Widdop, B.K. Kemp-
Harper, Nitroxyl (HNO): the cinderella of the nitric oxide story, Trends
Pharmacol. Sci. 29 (2008) 601–608.
hydroxybenzenecarboximidic acid derivatives:
a new class of nitroxyl-
generating prodrugs, Nitric Oxide 5 (2001) 278–287.
[5] B.K. Kemp-Harper, Nitroxyl (HNO): a novel redox signaling molecule, Antioxid.
Redox Signal. 14 (2011) 1609–1613.
[6] J.F. DuMond, S.B. King, The chemistry of nitroxyl-releasing compounds,
Antioxid. Redox Signal. 14 (2011) 1637–1648.
[26] D. Andrei, D.J. Salmon, S. Donzelli, A. Wahab, J.R. Klose, M.L. Citro, J.E. Saavedra,
D.A. Wink, K.M. Miranda, L.K. Keefer, Dual mechanisms of HNO generation by a
nitroxyl prodrug of the diazeniumdiolate (NONOate) class, J. Am. Chem. Soc.
132 (2010) 16526–16532.
[7] J.M. Fukuto, C.J. Cisneros, R.L. Kinkade,
A comparison of the chemistry
[27] G.A. Poskrebyshev, V. Shafirovich, S.V. Lymar, Hyponitrite radical, a stable
adduct of nitric oxide and nitroxyl, J. Am. Chem. Soc. 126 (2004) 891–899.
[28] S.V. Lymar, V. Shafirovich, G.A. Poskrebyshev, One-electron reduction of
aqueous nitric oxide: a mechanistic revision, Inorg. Chem. 44 (2005) 5212–
5221.
associated with the biological signaling and actions of nitroxyl (HNO) and
nitric oxide (NO), J. Inorg. Biochem. 118 (2013) 201–208.
[8] M.N. Hughes, P.E. Wimbledon, The chemistry of trioxodinitrates. Part 1.
Decomposition of sodium trioxodinitrate (Angeli’s salt) in aqueous solution, J.
Chem. Soc., Dalton Trans. (1976) 703–707.