an ioxidan s
A icle
EPR S udy o KO2as a Sou ce o Supe oxide and
•BMPO-OH/OOH Radical Tha Clea es Plasmid DNA and
De ec s Radical In e ac ion wi h H2S and Se-De i a i es
An on Misak 1, Vlas a B ezo a 2, Mi osla Cho anec 3, Ka ol Luspai 2, Muhammad Jawad Nasim 4,
Ma ian G man 1, Lenka Tomaso a 1, Claus Jacob 4and Ka ol Ond ias 1,*


Ci a ion: Misak, A.; B ezo a, V.;
Cho anec, M.; Luspai, K.; Nasim,
M.J.; G man, M.; Tomaso a, L.; Jacob,
C.; Ond ias, K. EPR S udy o KO2as a
Sou ce o Supe oxide and
•BMPO-OH/OOH Radical Tha
Clea es Plasmid DNA and De ec s
Radical In e ac ion wi h H2S and
Se-De i a i es. An ioxidan s 2021,10,
1286. h ps://doi.o g/10.3390/
an iox10081286
Academic Edi o s: Kenne h R. Olson
and Bulen Mu us
Recei ed: 25 June 2021
Accep ed: 11 Augus 2021
Published: 13 Augus 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Biomedical Resea ch Cen e , Depa men o Molecula Physiology, Ins i u e o Clinical and T ansla ional
Resea ch, Slo ak Academy o Sciences, Dúb a skáCes a 9, 84505 B a isla a, Slo akia;
[email p o ec ed] (A.M.); [email p o ec ed] (M.G.); [email p o ec ed] (L.T.)
2Ins i u e o Physical Chemis y and Chemical Physics, Facul y o Chemical and Food Technology,
Slo ak Uni e si y o Technology in B a isla a, Radlinského 9, 81237 B a isla a, Slo akia;
[email p o ec ed] (V.B.); ka [email p o ec ed] (K.L.)
3
Biomedical Resea ch Cen e , Depa men o Gene ics, Cance Resea ch Ins i u e, Slo ak Academy o Sciences,
Dúb a skáCes a 9, 84505 B a isla a, Slo akia; mi osla [email p o ec ed]
4Di ision o Bioo ganic Chemis y, School o Pha macy, Uni e si y o Saa land,
D-66123 Saa b uecken, Ge many; [email p o ec ed] (M.J.N.); [email p o ec ed] (C.J.)
*Co espondence: ka [email p o ec ed]
Abs ac :
Supe oxide adical anion (O
2•−
) and i s de i a i es egula e nume ous physiological and
pa hological p ocesses, which a e ex ensi ely s udied. The aim o ou wo k was o u ilize KO
2
as a
sou ce o O
2•−
and he elec on pa amagne ic esonance (EPR) spin apping 5- e -bu oxyca bonyl-5-
me hyl-1-py oline N-oxide (BMPO) echnique o he p epa a ion o
•
BMPO-OOH and/o
•
BMPO-
OH adicals in wa e solu ion wi hou DMSO. The me hod dis inguishes he in e ac ions o a ious
compounds wi h
•
BMPO-OOH and/o
•
BMPO-OH adicals o e ime. He e, we show ha he
addi ion o a bu e ed BMPO-HCl mix u e o powde ed KO
2
o med ela i ely s able
•
BMPO-OOH
and
•
BMPO-OH adicals and H
2
O
2
, whe e he
•
BMPO-OOH/OH a io depended on he pH. A a
inal pH o ~6.5–8.0, he concen a ion o
•
BMPO-OOH adicals was
≥
20 imes highe han ha o
•
BMPO-OH, whe eas a pH 9.0–10.0, he
•
BMPO-OH adicals p e ailed. The
•
BMPO-OOH/OH
adicals e ec i ely clea ed he plasmid DNA. H
2
S dec eased he concen a ion o
•
BMPO-OOH/OH
adicals, whe eas he selenium de i a i es 1-me hyl-4-(3-(phenylselanyl) p opyl) pipe azine and
1-me hyl-4-(4-(phenylselanyl) bu yl) pipe azine inc eased he p opo ion o
•
BMPO-OH o e he
•
BMPO-OOH adicals. In conclusion, he p esen ed app oach o using KO
2
as a sou ce o O
2•−
/H
2
O
2
and EPR spin ap BMPO o he p epa a ion o
•
BMPO-OOH/OH adicals in a physiological solu ion
could be use ul o s udy he biological e ec s o adicals and hei in e ac ions wi h compounds.
Keywo ds:
KO
2
; an ioxidan s; EPR spec a simula ion;
•
BMPO-OOH spin adduc ; supe oxide;
adical; hyd ogen sul ide; selenium-de i a i es; clea age DNA
1. In oduc ion
Supe oxide anion adical (O
2•−
) is essen ial o he li e o ae obic o ganisms, egula ing
nume ous physiological and pa hophysiological p ocesses. When an o e p oduc ion o
O
2•−
occu s and/o he an ioxidan de ense is de icien , oxida i e s ess may de elop,
leading o oxida i e damage in many biological sys ems [
1
,
2
]. The e o e, he p oduc ion,
biological in e ac ions, and con ol o he O
2•−
concen a ion ha e all been in ensi ely
s udied bo h in i o and in i o.
Fo expe imen s in ol ing adical O
2•−
eac ions, O
2•−
can be gene a ed using a i-
ous me hods [
3
], e.g., enzyma ically by xan hine/xan hine oxidase [
4
,
5
], which is a sys em
oo complex o some ypes o O
2•−
adical models. I can also be gene a ed by ionizing
An ioxidan s 2021,10, 1286. h ps://doi.o g/10.3390/an iox10081286 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2021,10, 1286 2 o 22
adia ion o by dissol ing supe oxide sal s in ap o ic sol en s, e.g., KO
2
in anhyd ous
DMSO; howe e , DMSO may no be desi able in some biological expe imen s [
3
,
6
]. On
he o he hand, KO
2
dissol ed in an aqueous solu ion is a simple sou ce o O
2•−
, bu i s
li e ime is in he ange o milliseconds [
7
]. Fo hese easons, i would be p ac ical and
app op ia e o ha e an easily accessible O
2•−
sou ce ha could be used in physiological
solu ions. The e o e, he aim o ou wo k was o examine KO
2
as a sui able sou ce o O
2•−
,
which can be used o eac wi h cyclic ni one 5- e -bu oxyca bonyl-5-me hyl-1-py oline-
N-oxide (BMPO), gene a ing supe oxide (
•
BMPO-OOH) o hyd oxyl (
•
BMPO-OH) adical
adduc s o u he s udies in aqueous sys ems.
The elec on pa amagne ic esonance (EPR) spin apping echnique, using BMPO,
is use ul o s udy sho -li ed O
2•−
and hyd oxyl (HO
•
) adicals [
4
,
5
,
8
–
15
]. By apping
O
2•−
, BMPO p oduces ela i ely s able
•
BMPO-OOH spin adduc s, which can be u he
s udied as a po en ial model o o ganic hyd ope oxides. A sa u a ed KO
2
solu ion in
DMSO has been used as a sou ce o O
2•−
o o m
•
BMPO-OOH. The p esence o DMSO
is con enien in s udies wi h wa e -insoluble compounds, bu i could be a limi a ion o
biological s udies.
Exogenously added and endogenously p oduced hyd ogen sul ide (H
2
S) in luences
many physiological and pa hologic p ocesses, including oxida i e s ess, by eac ing wi h
eac i e oxygen species [
16
,
17
]. O ganoselenium compounds ha e been known o se e
as mul i unc ional agen s. Such compounds ha e been shown o p o ide an excellen
an i-cance ac i i y by se ing as p o-oxidan s ha ul ima ely al e he edox homeos a-
sis o cance cells, leading o apop osis. Many biological ac i i ies ha e been associa ed
wi h o ganoselenium compounds, anging om simple an ioxidan s and an imic obials,
o a he complica ed immuno-modula o y, an i-in lamma o y, and an i-nocicep i e e -
ec s [
18
]. As o ganoselenium compounds ha e also been known o hei in e ac ion wi h
adicals, he compounds RSe-1 and RSe-2 we e employed in his s udy.
The e o e, in he p esen wo k, a new p ocedu e o he p epa a ion o ela i e s able
•
BMPO-OOH/OH adduc s om KO
2
as a sou ce o O
2•−
wi hou he use o DMSO as
a sol en is desc ibed. The
•
BMPO-OOH adical was mos ly obse ed a pH ~6.5–8.0.
•
BMPO-OOH/OH clea ed plasmid DNA and
•
BMPO-OOH/
•
BMPO-OH o al adical
concen a ions and hei p opo ion we e in luenced by H
2
S- and selenium-con aining
o ganic compounds.
2. Ma e ials and Me hods
2.1. Chemicals, EPR Sample P epa a ion, and Measu emen
Na
2
S was pu chased om DoJindo (SB01, Munich, Ge many), and he o he chemicals
we e pu chased om Sigma-Ald ich (S einheim, Ge many). Na
2
S dissocia es in he solu ion
and eac s wi h H
+
o yield H
2
S, HS
−
, and aces o S
2−
. We used H
2
S o e e he o al
mix u e o H
2
S, HS
−
, and S
2−
. A s ock solu ion o 100 mmol L
−1
Na
2
S was p epa ed in
a gon-bubbled ul apu e deionized H
2
O, and was s o ed a
−
80
◦
C o a ew days and
used immedia ely a e hawing.
Two selenium de i a i es, 1-me hyl-4-(3-(phenylselanyl)p opyl)pipe azine (RSe-1)
and 1-me hyl-4-(4-(phenylselanyl)bu yl)pipe azine (RSe-2), we e ob ained acco ding o he
syn hesis ou es shown in Scheme 1; Scheme 2. Fo he compound RSe-1, me hyl pipe azine
was alkyla ed, employing 1-b omo-3-chlo op opane in he p esence o anhyd ous K2CO3
and ace one as a sol en . The eac ion was pe o med a oom empe a u e (RT) and he
p og ess o he eac ion was con olled by hin laye ch oma og aphy. The c ude p oduc
was used o Se-alkyla ion in absolu e e hanol unde a ni ogen a mosphe e a oom
empe a u e (Scheme 1). Fo he compound RSe-2, Se-alkyla ion o 1-b omo-4-chlo obu ane
was pe o med unde a ni ogen a mosphe e and in absolu e e hanol a RT. The c ude
p oduc was hen eac ed wi h me hyl pipe azine in he p esence o anhyd ous K
2
CO
3
and ace one a 50
◦
C (Scheme 2) [
19
]. The s uc u e and pu i y o he inal compounds
we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p oduc s we e
con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e .
An ioxidan s 2021,10, 1286 3 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 3 o 23
K2CO3 and ace one a 50 °C (Scheme 2) [19]. The s uc u e and pu i y o he inal com-
pounds we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p od-
uc s we e con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e .
Scheme 1. Syn hesis o compound RSe-1. (a)The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine wi h
1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K2CO3 and ace one a oom
empe a u e. (b) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine en-
i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc .
Scheme 2. Syn hesis o compound RSe-2. (a)The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu-
ane wi h diphenyl diselenide in an ine en i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom
empe a u e o ob ain (4-chlo obu yl)(phenyl)selane. (b) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h
1-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc .
The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide
(BMPO, DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H2O (100
mmol L−1), s o ed a −80 °C, and used a e hawing. Fo he EPR samples, a buffe con-
sis ing o 50 mmol L−1 sodium phospha e and 100 µmol L−1 die hylene iaminepen aace ic
acid (DTPA; pH 7.4; 37 °C) was used. Powde ed KO2 (Sigma-Ald ich 278904, S einheim,
Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he
dissolu ion o KO2 in a phospha e bu e shi s om pH o alkaline alues, an adequa e
olume o 1 mol L−1 HCl was added o he bu e con aining BMPO (20 mmol L−1) o ge
he equi ed inal pH a e he addi ion o KO2. Twel e seconds a e adding he BMPO-
con aining phospha e solu ion o he powde ed KO2, compounds Na2S in H2O and RSe-1
o RSe-2, p epa ed in he bu e , we e added. The pH in he samples was measu ed by pH
indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy o ±0.2. The de ailed p o-
cedu e o he sample p epa a ion o EPR measu emen is desc ibed in he Supplemen-
a y Ma e ials. The sample was ans e ed o a s anda d ca i y aqueous EPR la cell (WG
808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he EPR spec a we e measu ed as de-
sc ibed p e iously [6]. The i s EPR spec um was eco ded 100 ± 15 s a e he addi ion
o he BMPO solu ion o powde ed KO2. The se s o indi idual EPR spec a o he BMPO
Scheme 1.
Syn hesis o compound RSe-1. (
a
)The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine
wi h 1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K
2
CO
3
and ace one a
oom empe a u e. (
b
) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine
en i onmen o N2, in he p esence o NaBH4and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc .
An ioxidan s 2021, 10, x FOR PEER REVIEW 3 o 23
K2CO3 and ace one a 50 °C (Scheme 2) [19]. The s uc u e and pu i y o he inal com-
pounds we e con i med ia spec oscopic and ch oma og aphic me hods. The inal p od-
uc s we e con e ed in o c ys alline hyd ochlo ic sal s o imp o e hei solubili y in wa e .
Scheme 1. Syn hesis o compound RSe-1. (a)The i s s ep o syn hesis in ol es he alkyla ion o 1-me hylpipe azine wi h
1-b omo-3-chl op opane o p oduce 1-(3-chlo op opyl)-4-me hylpipe azine in he p esence o K2CO3 and ace one a oom
empe a u e. (b) 1-(3-chlo op opyl)-4-me hylpipe azine is subsequen ly eac ed wi h diphenyl diselenide in an ine en-
i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom empe a u e o ob ain RSe-1 as inal p oduc .
Scheme 2. Syn hesis o compound RSe-2. (a)The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu-
ane wi h diphenyl diselenide in an ine en i onmen o N2, in he p esence o NaBH4 and absolu e e hanol a oom
empe a u e o ob ain (4-chlo obu yl)(phenyl)selane. (b) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h
1-me hylpipe azine in he p esence o K2CO3 and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc .
The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide
(BMPO, DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H2O (100
mmol L−1), s o ed a −80 °C, and used a e hawing. Fo he EPR samples, a buffe con-
sis ing o 50 mmol L−1 sodium phospha e and 100 µmol L−1 die hylene iaminepen aace ic
acid (DTPA; pH 7.4; 37 °C) was used. Powde ed KO2 (Sigma-Ald ich 278904, S einheim,
Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he
dissolu ion o KO2 in a phospha e bu e shi s om pH o alkaline alues, an adequa e
olume o 1 mol L−1 HCl was added o he bu e con aining BMPO (20 mmol L−1) o ge
he equi ed inal pH a e he addi ion o KO2. Twel e seconds a e adding he BMPO-
con aining phospha e solu ion o he powde ed KO2, compounds Na2S in H2O and RSe-1
o RSe-2, p epa ed in he bu e , we e added. The pH in he samples was measu ed by pH
indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy o ±0.2. The de ailed p o-
cedu e o he sample p epa a ion o EPR measu emen is desc ibed in he Supplemen-
a y Ma e ials. The sample was ans e ed o a s anda d ca i y aqueous EPR la cell (WG
808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he EPR spec a we e measu ed as de-
sc ibed p e iously [6]. The i s EPR spec um was eco ded 100 ± 15 s a e he addi ion
o he BMPO solu ion o powde ed KO2. The se s o indi idual EPR spec a o he BMPO
Scheme 2.
Syn hesis o compound RSe-2. (
a
)The i s s ep o syn hesis in ol es he Se-alkyla ion o 1-b omo-4-chlo obu ane
wi h diphenyl diselenide in an ine en i onmen o N
2
, in he p esence o NaBH
4
and absolu e e hanol a oom em-
pe a u e o ob ain (4-chlo obu yl)(phenyl)selane. (
b
) In he second s ep (4-chlo obu yl)(phenyl)selane is eac ed wi h
1-me hylpipe azine in he p esence o K2CO3and ace one a oom empe a u e o ob ain RSe-2 as inal p oduc .
The spin- apping agen , 5- e -bu oxyca bonyl-5-me hyl-1-py oline N-oxide (BMPO,
DoJindo B568, Munich, Ge many), was dissol ed in ul apu e deionized H
2
O (
100 mmol L−1
),
s o ed a
−
80
◦
C, and used a e hawing. Fo he EPR samples, a bu e consis ing o
50 mmol L
−1
sodium phospha e and 100
µ
mol L
−1
die hylene iaminepen aace ic acid
(DTPA; pH 7.4; 37
◦
C) was used. Powde ed KO
2
(Sigma-Ald ich 278904, S einheim,
Ge many) in amoun s o 0.5–1 mg was weighed in o ubes jus be o e being used. As he
dissolu ion o KO
2
in a phospha e bu e shi s om pH o alkaline alues, an adequa e
olume o 1 mol L
−1
HCl was added o he bu e con aining BMPO (20 mmol L
−1
)
o ge he equi ed inal pH a e he addi ion o KO
2
. Twel e seconds a e adding
he BMPO-con aining phospha e solu ion o he powde ed KO
2
, compounds Na
2
S in
H
2
O and RSe-1 o RSe-2, p epa ed in he bu e , we e added. The pH in he samples
was measu ed by pH indica o pape (Ahls om-Munksjö, Ge many) wi h an accu acy
o
±
0.2. The de ailed p ocedu e o he sample p epa a ion o EPR measu emen is
desc ibed in he Supplemen a y Ma e ials. The sample was ans e ed o a s anda d
ca i y aqueous EPR la cell (WG 808-Q, Wilmad-LabGlass, Vineland, NJ, USA), and he
EPR spec a we e measu ed as desc ibed p e iously [
6
]. The i s EPR spec um was
eco ded 100
±
15 s a e he addi ion o he BMPO solu ion o powde ed KO
2
. The
se s o indi idual EPR spec a o he BMPO spin adduc s we e eco ded as 15 o 30
sequen ial scans o 42 s each, wi h a o al acquisi ion ime o 11 o 22 min, espec i ely. The
An ioxidan s 2021,10, 1286 4 o 22
EPR spec a o he BMPO spin adduc s we e measu ed on a B uke EMX spec ome e
(Rheins e en, Ge many) essen ially as in ou p e ious s udy [
6
]. All o he EPR spec a
we e eco ded a 37
◦
C. The spec a we e simula ed using he EasySpin p og am wo king
on he Ma Lab pla o m [
20
]. The concen a ion o
•
BMPO-OOH and
•
BMPO-OH adicals
was e alua ed om a double in eg al o hei EPR spec a and compa ed wi h he spec a
o 20
µ
mol L
−1
4-hyd oxy-2,2,6,6- e ame hylpipe idine-1-oxyl adical (TEMPOL, Fluka
42777, Munich, Ge many), which was used as a e e ence sample. To compa e he po ency
o he in es iga ed compounds o dec easing he o e all apped adical concen a ion,
a double in eg al o he o al EPR spec a in ensi y o he BMPO adduc s was e alua ed.
The concen a ion o H
2
O
2
was measu ed by a Fluo ime ic Hyd ogen Pe oxide Assay
Ki (MAK165, Me ck, B a isla a, Slo akia) acco ding o he manu ac u e ’s ins uc ions.
The concen a ions o KO
2
and he BMPO/KO
2
mix u e we e dilu ed 1000 and 2000 imes,
espec i ely, be o e he measu emen o H2O2.
2.2. Plasmid DNA (pDNA) Clea age Assay
A pDNA clea age assay wi h he use o he pBR322 plasmid (N3033 L, New England
BioLabs, Inc., Ipswich, MA, USA) was pe o med as epo ed p e iously, wi h some
modi ica ions [
6
]. All o he inal samples con ained he s udied compounds and 0.2
µ
g
o pDNA in 20
µ
L o he sodium phospha e bu e (25 mmol L
−1
sodium phospha e,
50 µmol L−1
DTPA; pH 6.5, 7.4, 8.0, o 8.5). The p ocedu e o he sample p epa a ion o
he pDNA clea age assay is desc ibed in de ail in he Supplemen a y Ma e ials. Likewise,
in he EPR sample p epa a ion, he phospha e bu e was adjus ed by HCl o keep he inal
pH a he equi ed alue a e being added o powde ed KO
2
(20 mmol L
−1
) (see P ocedu e
5 in Supplemen a y Ma e ials). In he p ocedu e ocused on he KO
2−
BMPO in e ac ion,
he powde ed KO
2
(20 mmol L
−1
) was dissol ed by 10 mmol L
−1
BMPO in a 45 mmol L
−1
phospha e bu e adjus ed by HCl, hen o exed o 10 s, and 10
µ
L o he mix u e was
added o a 10
µ
L solu ion o pDNA in a 5 mmol L
−1
phospha e bu e (see P ocedu e 6
in Supplemen a y Ma e ials). The pH condi ions we e also s anda dized in he clea age
assay h ough he addi ion o 0.5 mol L
−1
NaOH ( o pH 6.5) o 0.1/0.125 mol L
−1
HCl
( o pH 8.0/8.5) o he pDNA solu ion o each pH 7.4 o all o he samples (see P ocedu e
7 in Supplemen a y Ma e ials).
Fo he p epa a ion o he BMPO/KO
2
mix u e, in which, acco ding o EPR expe i-
men s,
•
BMPO-OOH/OH adicals we e no o med, powde ed KO
2
(20 mmol L
−1
) was
dissol ed in a 50 mmol L
−1
phospha e bu e adjus ed by HCl, o exed o 10 s, and hen
BMPO (10 mmol L
−1
) was added. As in he p e ious expe imen s, 10
µ
L o he mix u e
was added o 10
µ
L o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials).
All o he inal samples con ained 5 mmol L
−1
BMPO and 10 mmol L
−1
KO
2
in he sodium
phospha e bu e (25 mmol L
−1
sodium phospha e, 50
µ
mol L
−1
DTPA; pH 6.5, 7.4, 8.0 o
8.5). FeCl
2
(150
µ
mol L
−1
), as a massi e pDNA clea age induc o , was used as a posi i e
con ol in he assay. The esul ing mix u es we e incuba ed o 30 min a 37
◦
C. A e
incuba ion, he eac ion mix u es we e subjec ed o 0.6% aga ose gel elec opho esis. The
in eg a ed densi ies o all pBR322 o ms in each lane we e quan i ied using Image S udio
analysis so wa e (LI-COR Bio echnology, Bad Hombu g, Ge many) o es ima e he pDNA
clea age po ency h ough he ela i e in ensi ies (I
R
) o he nicked o m o pDNA (see
Figu e S2 in Supplemen a y Ma e ials).
3. Resul s
3.1. pH-Dependen Composi ion o •BMPO-Adduc s o (BMPO+HCl)-KO2In e ac ion
In he con ol expe imen , a phospha e bu e adjus ed wi h HCl was added o pow-
de ed KO
2
( inal 40 mmol L
−1
KO
2
) o ge a inal pH alue o 7.4. A e 10–13 s, when
BMPO ( inal 20 mmol L
−1
) was added o he KO
2
–bu e solu ion, he EPR spec a o
•
BMPO adduc s we e no obse ed ( o de ails, see P ocedu e 1 in Supplemen a y Ma e i-
als). Howe e , when BMPO (20 mmol L
−1
) in a phospha e bu e (pH 7.4) was added o
powde ed KO
2
( inal 40 mmol L
−1
), EPR spec a, depending on inal pH, we e obse ed
An ioxidan s 2021,10, 1286 5 o 22
(Figu e 1). A e he BMPO-con aining phospha e solu ion (pH 7.4) was added o he
powde ed KO
2
, he pH inc eased o ~10–11. The e o e, o s udy he pH dependence o
he adical o ma ion om KO
2
, HCl was added o BMPO in he bu e , which ensu ed
ha a e he BMPO solu ion was added o he powde ed KO
2
, he inal equi ed pH
was ob ained ( o de ails, see P ocedu e 2 in Supplemen a y Ma e ials). The in ensi y
and shapes o he
•
BMPO adduc s spec a depended on he pH. The spec al in ensi y
o
•
BMPO adduc s ob ained a pH 9.0 dec eased in ime (Figu e 1a1–a3). In compa ison
wi h pH 9.0, he EPR spec al in ensi y o he
•
BMPO adduc s a pH 7.7 was highe wi h
a di e en ela i e abundance o indi idual species and slow decay o he adduc s. The
•
BMPO adduc s we e obse ed o mo e han 22 min a pH 7.7 (Figu e 1b1–c3). A simila
e ec on he spec a o he
•
BMPO adduc s was also obse ed a pH 6.5 (Figu e 1d1–e3)
and pH 2 (Figu e 1 1–g3). By compa ison, he in ensi y o he EPR spec a o he
•
BMPO
adduc s o med in he mix u e o 5 mmol L
−1
BMPO and 10 mmol L
−1
KO
2
a pH 7.4
was quan i a i ely lowe (Figu e 1h1–h3), bu simila o ha o he highe BMPO–KO
2
concen a ion (Figu e 1b1–e3). The s able ni oxide adical TEMPOL (20
µ
mol L
−1
) was
used o de e mine he concen a ion o he adicals in he samples (Figu e 1i1–i3). Using
he double in eg als o he TEMPOL calib a ion, he concen a ion o he o al
•
BMPO
adduc adicals was high a pH 6.5–7.8 and low a pH 9.0–11.5, and dec eased in ime, wi h
a hal ime (pH 6.5–7.8) o app oxima ely ~12 min (Figu e 2a).
As desc ibed in de ail in Sec ion 3.2, he i s eco ded spec um was simula ed o
ob ain he indi idual componen s o
•
BMPO-OOH (sum o con o me s 1 and 2) and
•
BMPO-OH (sum o con o me s 1 and 2) a 100
±
15 s a e he sample p epa a ion
(Figu e 2b). The a io o he componen popula ion signi ican ly depended on he pH; he
po ions o
•
BMPO-OOH and
•
BMPO-OH we e ~95% and ~5%, espec i ely, o he whole
concen a ion wi hin a pH o ~2–8, and ~10% and ~90% wi hin a pH o ~9–11.
As H
2
O
2
is p oduced du ing he eac ion o KO
2
wi h H
2
O, i s concen a ion was
measu ed in samples con aining KO
2
a pH 7.4. H
2
O
2
concen a ions o 8.9
±
1.4 (n= 4)
and 10.5
±
0.7 (n= 4) mmol L
−1
we e o med a e he addi ion o bu e + HCl o BMPO +
HCl o he powde ed KO2( inal 20/40 BMPO/KO2in mmol L−1), espec i ely.
3.2. Simula ion o EPR Spec a o pH-Dependen BMPO Adduc s
The shapes o he EPR spec a o he BMPO adduc s depended on he pH (Figu e 1)
and changed o e ime, indica ing a dynamic supe posi ion o signals co esponding o
he gene a ion o indi idual BMPO adduc s. The e o e, we analyzed he EPR spec a by
simula ion, as in [
6
]. Two con o me s each o
•
BMPO-OOH and
•
BMPO-OH adduc s we e
inse ed in o he spin Hamil onian calcula ions o he expe imen al spec a measu ed in
he pH ange o 2–8. Howe e , in he EPR spec a measu ed in he solu ions a pH
≥
9, wo
addi ional low-in ensi y six-line signals we e de ec ed, which we e assigned o he BMPO
adduc wi h a ca bon-cen e ed adical (
•
BMPO-CR) [
8
] and
•
BMPO-CO
2−
, mos likely
o igina ing om he DTPA decomposi ion in he alkaline solu ions. The simula ed spec a
shown in Figu e 3we e calcula ed using he hype ine coupling cons an s (h cc) elucida ed
om he expe imen al spec a (Table 1). The compa ison o he expe imen al and simula ed
spec a shows a good i when using he h cc. The de ailed simula ion analysis o he h cc
o he indi idual BMPO adduc s e alua ed om he EPR spec a measu ed a a ious pH
alues e ealed only a negligible pH e ec on he h cc.

An ioxidan s 2021,10, 1286 6 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 6 o 23
Figu e 1. Rep esen a i e pH-dependen EPR spec a o •BMPO adduc s a e he addi ion o 20
mmol L–1 BMPO + HCl o powde ed KO2 ( inal 40 mmol L–1 KO2). (a1–i1) Collec ion o 15 EPR spec-
a o BMPO adduc s a anged back- o-back, each 42 s, wi h a s a ing acquisi ion o 100 ± 15 s a e
sample p epa a ion. (a2–i2) The i s o i h accumula ed spec a and (a3–i3) las i e accumula ed
spec a. HCl was added o BMPO (20 mmol L–1) in a bu e o he inal equi ed pH and he solu ion
was added o powde ed KO2. (a1–a3) pH 9.0; (b1–b3) pH 7.7 and (c1–c3) con inua ion; (d1–d3) pH
6.5 and (e1–e3) con inua ion; ( 1– 3) pH 2.0 and (g1–g3) con inua ion. (h1–h3) •BMPO adduc s a e
he addi ion o 5 mmol L–1 BMPO + HCl o powde ed KO2 ( inal 10 mM KO2) a pH 7.4. (i1–i3) The
EPR spec a o s able adical TEMPOL (20 µmol L–1). In ensi ies o (a1–h1) ime-dependen EPR
spec a and (a2–h3) he de ailed spec a a e app oxima ely compa able. Fo de ails o he sample
p epa a ion, see P ocedu e 2 in Supplemen a y Ma e ials.
Figu e 1.
Rep esen a i e pH-dependen EPR spec a o
•
BMPO adduc s a e he addi ion o 20 mmol
L
−1
BMPO + HCl o powde ed KO
2
( inal 40 mmol L
−1
KO
2
). (
a1
–
i1
) Collec ion o 15 EPR spec a o
BMPO adduc s a anged back- o-back, each 42 s, wi h a s a ing acquisi ion o 100
±
15 s a e sample
p epa a ion. (
a2
–
i2
) The i s o i h accumula ed spec a and (
a3
–
i3
) las i e accumula ed spec a.
HCl was added o BMPO (20 mmol L
−1
) in a bu e o he inal equi ed pH and he solu ion was
added o powde ed KO
2
. (
a1
–
a3
) pH 9.0; (
b1
–
b3
) pH 7.7 and (
c1
–
c3
) con inua ion; (
d1
–
d3
) pH 6.5
and (
e1
–
e3
) con inua ion; (
1
–
3
) pH 2.0 and (
g1
–
g3
) con inua ion. (
h1
–
h3
)
•
BMPO adduc s a e he
addi ion o 5 mmol L
−1
BMPO + HCl o powde ed KO
2
( inal 10 mM KO
2
) a pH 7.4. (
i1
–
i3
) The
EPR spec a o s able adical TEMPOL (20
µ
mol L
−1
). In ensi ies o (
a1
–
h1
) ime-dependen EPR
spec a and (
a2
–
h3
) he de ailed spec a a e app oxima ely compa able. Fo de ails o he sample
p epa a ion, see P ocedu e 2 in Supplemen a y Ma e ials.
An ioxidan s 2021,10, 1286 7 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 7 o 23
Figu e 2. (a) Time and pH dependencies o he o al •BMPO adduc adical concen a ion o he
BMPO + HCl/KO2 (20/40 in mmol L−1) mix u e. Each poin ep esen s he a e age o i e accumu-
la ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO + HCl o powde ed KO2. (b)
pH-dependen no malized in eg al EPR in ensi y o indi idual •BMPO-OOH and •BMPO-OH com-
ponen s elucida ed om he simula ion o he i s expe imen al EPR spec um eco ded 100 ± 15 s
a e he addi ion o BMPO + HCl o powde ed KO2. Spec al simula ion is desc ibed in Sec ion 3.2.
Spec al componen s: •BMPO-OOH (blue) and •BMPO-OH ( ed). The sum o indi idual BMPO ad-
duc s o he i s spec a was no malized o 100%. Each pai (blue and ed) o wo complemen a y
poin s ep esen s he indi idual sample p epa a ion.
As desc ibed in de ail in Sec ion 3.2, he i s eco ded spec um was simula ed o
ob ain he indi idual componen s o •BMPO-OOH (sum o con o me s 1 and 2) and
•BMPO-OH (sum o con o me s 1 and 2) a 100 ± 15 s a e he sample p epa a ion (Figu e
2b). The a io o he componen popula ion signi ican ly depended on he pH; he po ions
o •BMPO-OOH and •BMPO-OH we e ~95% and ~5%, espec i ely, o he whole concen-
a ion wi hin a pH o ~2–8, and ~10% and ~90% wi hin a pH o ~9–11.
As H2O2 is p oduced du ing he eac ion o KO2 wi h H2O, i s concen a ion was
measu ed in samples con aining KO2 a pH 7.4. H2O2 concen a ions o 8.9 ± 1.4 (n = 4) and
10.5 ± 0.7 (n = 4) mmol L–1 we e o med a e he addi ion o bu e + HCl o BMPO + HCl
o he powde ed KO2 ( inal 20/40 BMPO/KO2 in mmol L–1), espec i ely.
3.2. Simula ion o EPR Spec a o pH-Dependen BMPO Adduc s
The shapes o he EPR spec a o he BMPO adduc s depended on he pH (Figu e 1)
and changed o e ime, indica ing a dynamic supe posi ion o signals co esponding o
Figu e 2.
(
a
) Time and pH dependencies o he o al
•
BMPO adduc adical concen a ion o he
BMPO + HCl/KO
2
(20/40 in mmol L
−1
) mix u e. Each poin ep esen s he a e age o i e accumu-
la ed subsequen EPR spec a. Time s a s a e he addi ion o BMPO + HCl o powde ed KO
2
. (
b
)
pH-dependen no malized in eg al EPR in ensi y o indi idual
•
BMPO-OOH and
•
BMPO-OH com-
ponen s elucida ed om he simula ion o he i s expe imen al EPR spec um eco ded 100 ±15 s
a e he addi ion o BMPO + HCl o powde ed KO
2
. Spec al simula ion is desc ibed in Sec ion 3.2.
Spec al componen s:
•
BMPO-OOH (blue) and
•
BMPO-OH ( ed). The sum o indi idual BMPO
adduc s o he i s spec a was no malized o 100%. Each pai (blue and ed) o wo complemen a y
poin s ep esen s he indi idual sample p epa a ion.
Table 1.
The h cc o BMPO spin adduc s elucida ed om simula ions o expe imen al spec a
measu ed in phospha e bu e solu ions con aining KO
2
.
•
BMPO-OOH and
•
BMPO-OH we e
simula ed conside ing he p esence o wo con o me s.
BMPO-Adduc aN, mT aHβ, mT aHγ, mT
•BMPO-OH(1) 1.433 ±0.003 1.521 ±0.005 0.074 ±0.004
•BMPO-OH(2) 1.421 ±0.004 1.264 ±0.003 0.065 ±0.002
•BMPO-OOH(1) 1.341 ±0.003 1.198 ±0.004 –
•BMPO-OOH(2) 1.340 ±0.001 0.967 ±0.006 –
•BMPO-CR 1.515 ±0.008 2.077 ±0.024 –
•BMPO-CO2−1.490 ±0.003 1.710 ±0.012 –
An ioxidan s 2021,10, 1286 8 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 8 o 23
he gene a ion o indi idual BMPO adduc s. The e o e, we analyzed he EPR spec a by
simula ion, as in [6]. Two con o me s each o •BMPO-OOH and •BMPO-OH adduc s we e
inse ed in o he spin Hamil onian calcula ions o he expe imen al spec a measu ed in
he pH ange o 2–8. Howe e , in he EPR spec a measu ed in he solu ions a pH ≥ 9,
wo addi ional low-in ensi y six-line signals we e de ec ed, which we e assigned o he
BMPO adduc wi h a ca bon-cen e ed adical (•BMPO-CR) [8] and •BMPO-CO2–, mos
likely o igina ing om he DTPA decomposi ion in he alkaline solu ions. The simula ed
spec a shown in Figu e 3 we e calcula ed using he hype ine coupling cons an s (h cc)
elucida ed om he expe imen al spec a (Table 1). The compa ison o he expe imen al
and simula ed spec a shows a good i when using he h cc. The de ailed simula ion anal-
ysis o he h cc o he indi idual BMPO adduc s e alua ed om he EPR spec a measu ed
a a ious pH alues e ealed only a negligible pH e ec on he h cc.
Figu e 3. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei
simula ions using he h cc summa ized in Table 1. Only he expe imen al spec a o he six h o
en h accumula ed spec a a e shown (blue, measu ed 5.2–8.7 min a e sample p epa a ion); he
simula ed spec a a e ed. (a) pH 9.0, (b) pH 7.7, (c) con inued measu emen du ing 15.7–19.2 min,
and (d) pH 6.0.
Table 1. The h cc o BMPO spin adduc s elucida ed om simula ions o expe imen al spec a meas-
u ed in phospha e bu e solu ions con aining KO2. •BMPO-OOH and •BMPO-OH we e simula ed
conside ing he p esence o wo con o me s.
Figu e 3.
Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei
simula ions using he h cc summa ized in Table 1. Only he expe imen al spec a o he six h o en h
accumula ed spec a a e shown (blue, measu ed 5.2–8.7 min a e sample p epa a ion); he simula ed
spec a a e ed. (
a
) pH 9.0, (
b
) pH 7.7, (
c
) con inued measu emen du ing 15.7–19.2 min, and (
d
) pH 6.0.
3.3. Compa ison o pH-Dependen BMPO Adduc Spec a o BMPO−KO2In e ac ion
Fo simplici y, he ela i e concen a ions o wo con o me s,
•
BMPO-OH(1) and
•
BMPO-OH(2), we e summed and desc ibed as
•
BMPO-OH. Analogously,
•
BMPO-OOH(1)
and
•
BMPO-OOH(2) we e summed and desc ibed as
•
BMPO-OOH. As desc ibed in Sec-
ion 3.1, he pH-dependen o ma ion o indi idual
•
BMPO-OOH and
•
BMPO-OH compo-
nen s elucida ed om he simula ion o he i s expe imen al EPR spec um is shown in
Figu e 2b. The ime dependence o he EPR in ensi y o he simula ed componen s o 1–5,
6–10, and 11–15 accumula ed expe imen al BMPO adduc spec a a di e en pH alues is
shown in Figu e 4. The sum o indi idual
•
BMPO adduc s o he accumula ed spec a a a
gi en ime was no malized o 100%. A pH 11.5, he
•
BMPO-OH componen dec eased in
ime, and
•
BMPO-CR and
•
BMPO-CO
2−
componen s we e p esen
(Figu e 4a).
Simila ly,
mos ly
•
BMPO-OH, as well as a mino abundance o
•
BMPO-CR,
•
BMPO-CO
2−
, and
•
BMPO-OOH componen s, we e obse ed a pH 9.0 (Figu e 4b). Howe e , a a lowe pH
(
≤
8.0), he
•
BMPO-OOH componen p e ailed o e
•
BMPO-OH (Figu e 4c,d,e,g). The el-
a i e concen a ion o
•
BMPO-OOH dec eased and
•
BMPO-OH inc eased g adually o e
ime a pH 7.7–8.0 (Figu e 4c–e). The ela i e concen a ion o
•
BMPO-OOH in compa ison
wi h •BMPO-OH was high a pH 6.0 and ela i ely s able o o e 11 min (Figu e 4g,h).
An ioxidan s 2021,10, 1286 9 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 10 o 23
Figu e 4. Compa ison o ime and pH-dependen no malized in eg al EPR in ensi y o indi idual
BMPO adduc s elucida ed om he simula ion o expe imen al EPR spec a. Spec al componen s:
•BMPO-OOH (blue), •BMPO-OH ( ed), •BMPO-CR (black), •BMPO-CO2- (g een) a (a) pH 11.5 (pH
was adjus ed by NaOH), (b) pH 9.0, (c) pH 8.0, (d) pH 7.8, and (e) pH 7.7; ( ) con inued measu emen
o (e) o he nex 11 min; (g) pH 6.0 and (h) con inued measu emen o (g) o he nex 11 min. Each
poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time s a s a e he ad-
di ion o BMPO + HCl o powde ed KO2. The sum o indi idual BMPO adduc s o accumula ed
spec a was no malized o 100%. Fo de ails o he sample p epa a ion, see P ocedu e 2 in Supple-
men a y Ma e ials.
To compa e he pH dependence o he o al adicals o he •BMPO adduc compo-
nen s, double in eg al in ensi ies o he simula ed spec a we e applied (Figu e 5). The
concen a ion o apped adicals o he •BMPO adduc s was e y low a pH ~9.0–11.5
(Figu e 5a,b). Howe e , a pH ~6.0–8.0, he numbe o apped adicals inc eased ~3–10
imes, pa icula ly because o he •BMPO-OOH componen (Figu e 5c–h). The amoun o
•BMPO-OOH adical componen s was ~10 imes highe in compa ison wi h •BMPO-OH
a pH ~6.0–8.0, 3.5 min a e he sample p epa a ion (Figu e 5c–h). The p esence o he
•BMPO-OOH componen was s ill ~3 imes mo e abundan in compa ison wi h •BMPO-
OH a e 22 min a pH ~6.0 (Figu e 5h).
Figu e 4.
Compa ison o ime and pH-dependen no malized in eg al EPR in ensi y o indi idual
BMPO adduc s elucida ed om he simula ion o expe imen al EPR spec a. Spec al componen s:
•
BMPO-OOH (blue),
•
BMPO-OH ( ed),
•
BMPO-CR (black),
•
BMPO-CO
2-
(g een) a (
a
) pH 11.5
(pH was adjus ed by NaOH), (
b
) pH 9.0, (
c
) pH 8.0, (
d
) pH 7.8, and (
e
) pH 7.7; (
) con inued
measu emen o (
e
) o he nex 11 min; (
g
) pH 6.0 and (
h
) con inued measu emen o (
g
) o he
nex 11 min. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time
s a s a e he addi ion o BMPO + HCl o powde ed KO
2
. The sum o indi idual BMPO adduc s o
accumula ed spec a was no malized o 100%. Fo de ails o he sample p epa a ion, see P ocedu e 2
in Supplemen a y Ma e ials.
To compa e he pH dependence o he o al adicals o he
•
BMPO adduc componen s,
double in eg al in ensi ies o he simula ed spec a we e applied (Figu e 5). The concen a-
ion o apped adicals o he
•
BMPO adduc s was e y low a pH ~9.0–11.5 (Figu e 5a,b).
Howe e , a pH ~6.0–8.0, he numbe o apped adicals inc eased ~3–10 imes, pa icu-
la ly because o he
•
BMPO-OOH componen (Figu e 5c–h). The amoun o
•
BMPO-OOH
adical componen s was ~10 imes highe in compa ison wi h
•
BMPO-OH a pH ~6.0–8.0,
3.5 min a e he sample p epa a ion (Figu e 5c–h). The p esence o he
•
BMPO-OOH
componen was s ill ~3 imes mo e abundan in compa ison wi h
•
BMPO-OH a e 22 min
a pH ~6.0 (Figu e 5h).
An ioxidan s 2021,10, 1286 16 o 22
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Figu e 12. Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei
simula ions. A angemen o expe imen al and simula ed spec a is he same as in Figu e 3. EPR
spec a a (a) pH 10.3, (b) pH 8.5, (c) pH 7.3, and (d) pH 6.5. Sample p epa a ion: BMPO in he bu e
was added o powde ed KO2 and o exed, and 30 s la e HCl was added o adjus he inal pH o
he solu ion.
Figu e 12.
Rep esen a i e no malized expe imen al EPR spec a o BMPO adduc s along wi h hei
simula ions. A angemen o expe imen al and simula ed spec a is he same as in Figu e 3. EPR
spec a a (
a
) pH 10.3, (
b
) pH 8.5, (
c
) pH 7.3, and (
d
) pH 6.5. Sample p epa a ion: BMPO in he bu e
was added o powde ed KO
2
and o exed, and 30 s la e HCl was added o adjus he inal pH o
he solu ion.

An ioxidan s 2021,10, 1286 17 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 18 o 23
Figu e 13. (Le column) Compa ison o ime- and pH-dependen no malized in eg al EPR in ensi y
o indi idual BMPO adduc s elucida ed om he simula ion o he expe imen al EPR spec a. Spec-
al componen s: •BMPO-OOH (blue), •BMPO-OH ( ed), •BMPO-CR (black), and •BMPO-CO2−
(g een) a (a) pH 10.3, (b) pH 8.5, and (c) pH 7.3; (d) con inued measu emen o (c) o ano he 11
min; (e) pH 6.5. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a.
Time s a s a e he addi ion o BMPO o powde ed KO2. The sum o he indi idual BMPO adduc s
o he accumula ed spec a was no malized o 100%. (Righ column) ( –j) Compa ison o ime- and
pH-dependen absolu e in eg al EPR in ensi y ( o al quan i y o adicals in .u.) o indi idual BMPO
adduc s o no malized in eg al EPR in ensi y shown in he le column.
3.6. pH-Dependen Clea age o pDNA by BMPO–KO2 In e ac ion
To know whe he •BMPO adduc s ha e biological e ec s, he clea age o pDNA by
•BMPO-OOH/OH was e alua ed (Figu es 14 and S2). pDNA clea age was negligible in
he con ol bu e s a pH 6.5, 7.4, 8.0, and 8.5, bu signi ican ly inc eased in he p esence
o FeCl2, which is known o clea e DNA [21,22]. Besides he single-s anded clea age o
he supe coiled pDNA gene a ing he nicked ci cula o m, FeCl2 was no ably able o me-
dia e double-s anded clea age, c ea ing linea o m (Figu e S2 in Supplemen a y Ma e-
ials). The con ol samples con aining BMPO, H2O2, KO2, o he mix u e o H2O2 and KO2
did no induce pDNA clea age. Howe e , when BMPO + HCl was added o powde ed
KO2, p oducing •BMPO-OOH/OH, and hen applied o pDNA, signi ican pDNA clea -
age was obse ed a all o he pH alues s udied. The alues we e sca e ed, bu he pDNA
clea age po ency had a endency o inc ease wi h he inc easing pH alues. To con i m
he in ol emen o pH in he obse ed e ec , he solu ion o pDNA in he phospha e
Figu e 13.
(Le column) Compa ison o ime- and pH-dependen no malized in eg al EPR in ensi y
o indi idual BMPO adduc s elucida ed om he simula ion o he expe imen al EPR spec a. Spec al
componen s:
•
BMPO-OOH (blue),
•
BMPO-OH ( ed),
•
BMPO-CR (black), and
•
BMPO-CO
2−
(g een)
a (
a
) pH 10.3, (
b
) pH 8.5, and (
c
) pH 7.3; (
d
) con inued measu emen o (
c
) o ano he 11 min;
(
e
) pH 6.5. Each poin ep esen s he a e age o i e accumula ed subsequen EPR spec a. Time
s a s a e he addi ion o BMPO o powde ed KO
2
. The sum o he indi idual BMPO adduc s o
he accumula ed spec a was no malized o 100%. (Righ column) (
–
j
) Compa ison o ime- and
pH-dependen absolu e in eg al EPR in ensi y ( o al quan i y o adicals in .u.) o indi idual BMPO
adduc s o no malized in eg al EPR in ensi y shown in he le column.
An ioxidan s 2021,10, 1286 18 o 22
An ioxidan s 2021, 10, x FOR PEER REVIEW 19 o 23
bu e was adjus ed by NaOH o HCl o s anda dize he pH o 7.4 a e he addi ion o
(BMPO + HCl) + KO2 solu ions wi h pH 6.5, 8.0, and 8.5. A e he pH was adjus ed o 7.4,
he e ec s o he mix u es we e s ill signi ican , bu he pH end was los . Based on he
EPR measu emen o he adical concen a ion, pDNA was signi ican ly clea ed by
•BMPO-OOH/OH a a concen a ion o ~18 µmol L−1, whe e he concen a ion a io o
•BMPO-OOH o •BMPO-OH a he beginning o he expe imen s was ≥20. Taking in o
conside a ion he sho li espan o O2•− in an aqueous solu ion, we p epa ed a con ol sam-
ple ha , acco ding o he EPR s udy, did no o m adicals, as ollows: i s , powde ed
KO2 was dissol ed in a phospha e bu e adjus ed wi h HCl o ge a 20 mmol L–1 KO2 so-
lu ion wi h a co esponding pH, hen, 10 s la e BMPO ( inal 10 mmol L–1) was added, and
his mix u e was applied a 1:1 olume a io o pDNA solu ion. In acco dance wi h he
EPR da a, he mix u e p epa ed in his way had a negligible e ec on he pDNA clea age
a all o he pH le els used (Figu e 14).
Figu e 14. Po ency o compounds o induce pDNA clea age a pH 6.5 (g een), 7.4 ( ed), 8.0 (cyan),
and 8.5 pH (pink) in a 25 mmol L−1 sodium phospha e bu e and 50 µmol L−1 DTPA a 37 °C. Con ol
wi hou and wi h 150 µmol L–1 FeCl2; con ols wi h 5 mmol L–1 BMPO, 5 mmol L–1 H2O2, 10 mmol
L–1 KO2, and 5 mmol L–1 H2O2 + 10 mmol L–1 KO2. Sample (BMPO + HCL) + KO2: BMPO ( inal 5
mmol L–1) in a phospha e bu e adjus ed by HCl ( o ge equi ed pH o BMPO/KO2 mix u e) was
added o powde ed KO2 ( inal 10 mmol L–1), hen he mix u e was added o he pDNA solu ion (see
P ocedu e 6 in Supplemen a y Ma e ials). Sample (BMPO + HCL) + KO2 adjus ed o pH 7.4: solu-
ions we e p epa ed as o p e ious samples wi h a ious pH le els, bu HCl o NaOH we e added
o he pDNA solu ion o s anda dize he pH o 7.4 (see P ocedu e 7 in Supplemen a y Ma e ials).
Sample (Bu e + HCl) + KO2 + BMPO: phospha e bu e adjus ed by HCl was added o powde KO2
( inal 10 mmol L–1), ollowed by he addi ion o BMPO ( inal 5 mmol L–1) 10 s la e , and he mix u e
was applied o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials). The inal concen-
a ion o pDNA was 0.2 µg in 20 µL. IR o nc DNA o m ep esen s he ela i e in ensi y o he
nicked ci cula pDNA. Da a ep esen alues om indi idual samples. Ho izon al black ma ks in-
dica e means ± SD.
4. Discussion
O2•− and i s de i a i es a e in ol ed in adical signaling and, mo e signi ican ly, in
he de elopmen o diseases due o oxida i e s ess-media ed damage o he cellula s uc-
u es [1,2]. To s udy he molecula mechanism o i s biological ac ions, i is con enien o
ha e a simple and ep oducible sou ce o O2•−. The e a e se e al me hods o p oduce O2•−
Figu e 14.
Po ency o compounds o induce pDNA clea age a pH 6.5 (g een), 7.4 ( ed), 8.0 (cyan),
and 8.5 pH (pink) in a 25 mmol L
−1
sodium phospha e bu e and 50
µ
mol L
−1
DTPA a 37
◦
C.
Con ol wi hou and wi h 150
µ
mol L
−1
FeCl
2
; con ols wi h 5 mmol L
−1
BMPO, 5 mmol L
−1
H
2
O
2
,
10 mmol L
−1
KO
2
, and 5 mmol L
−1
H
2
O
2
+ 10 mmol L
−1
KO
2
. Sample (BMPO + HCL) + KO
2
:
BMPO ( inal 5 mmol L
−1
) in a phospha e bu e adjus ed by HCl ( o ge equi ed pH o BMPO/KO
2
mix u e) was added o powde ed KO
2
( inal 10 mmol L
−1
), hen he mix u e was added o he pDNA
solu ion (see P ocedu e 6 in Supplemen a y Ma e ials). Sample (BMPO + HCL) + KO
2
adjus ed o
pH 7.4: solu ions we e p epa ed as o p e ious samples wi h a ious pH le els, bu HCl o NaOH
we e added o he pDNA solu ion o s anda dize he pH o 7.4 (see P ocedu e 7 in Supplemen a y
Ma e ials). Sample (Bu e + HCl) + KO
2
+ BMPO: phospha e bu e adjus ed by HCl was added o
powde KO
2
( inal 10 mmol L
−1
), ollowed by he addi ion o BMPO ( inal 5 mmol L
−1
) 10 s la e ,
and he mix u e was applied o he pDNA solu ion (see P ocedu e 8 in Supplemen a y Ma e ials).
The inal concen a ion o pDNA was 0.2
µ
g in 20
µ
L. I
R
o nc DNA o m ep esen s he ela i e
in ensi y o he nicked ci cula pDNA. Da a ep esen alues om indi idual samples. Ho izon al
black ma ks indica e means ±SD.
4. Discussion
O
2•−
and i s de i a i es a e in ol ed in adical signaling and, mo e signi ican ly,
in he de elopmen o diseases due o oxida i e s ess-media ed damage o he cellula
s uc u es [
1
,
2
]. To s udy he molecula mechanism o i s biological ac ions, i is con enien
o ha e a simple and ep oducible sou ce o O
2•−
. The e a e se e al me hods o p oduce
O
2•−
[
3
]; howe e , some o hem a e no expe imen ally simple o a e no sui able o
biological expe imen s. The e o e, KO
2
as a sou ce o O
2•−
was s udied by he EPR
spin apping me hod wi hou using non-biological componen s. As in ou aqueous
expe imen al sys em he O
2•−
is highly eac i e and spon aneously disp opo iona es
wi hin 10 s, his s udy was limi ed mos ly o moni o ing he seconda y adicals (spin
adduc s)
•
BMPO-OOH/
•
BMPO-OH, and did no s udy he p oduc ion a es and a e o
any p ima y adicals. Addi ionally, we a e conscious o he pH-dependen eac i i y o
HO
2−
/H
2
O
2
p oduced in he O
2•−
disp opo iona ion in he p ocess o BMPO adduc
gene a ion.
The dissolu ion o KO
2
by a phospha e bu e p oduces O
2•−
and H
2
O
2
. O
2•−
is
known o o m
•
BMPO-OOH o
•
BMPO-OH adicals, which a e de ec able by EPR and
can be ob ained by spec al simula ion. F om he good i o he expe imen al spec a,
i is ob ious ha he h cc a e sui able o spec al simula ion. The h cc alues a e in he
An ioxidan s 2021,10, 1286 19 o 22
ange o da a ob ained unde simila expe imen al condi ions [
4
,
6
,
8
,
9
,
11
]. As ou me hod
dis inguishes concen a ions o he ime-dependen changes o
•
BMPO-OOH and
•
BMPO-
OH adicals in he same sample, i is app op ia e o ime-dependen s udies o compound
in e ac ions wi h adicals. In
•
BMPO-OOH and
•
BMPO-OH ni oxide (aminoxyl) adicals,
he unpai ed elec on is mos ly localized on he N-O moie y and he hype ine in e ac ions
wi h ni ogen and
β
-hyd ogen nuclei domina e he EPR spec a (Table 1). The e o e,
•
BMPO-OOH and
•
BMPO-OH can be u he s udied as po en ial models o o ganic
hyd ope oxide and alcohol, espec i ely.
The dissolu ion o KO
2
in a wa e solu ion (e.g., pH 7.4) p oduces O
2•−
wi h a sho
li e ime (<ms) [
3
,
7
,
23
]. This was con i med unde ou expe imen al condi ions a pH 7.4,
in which spin ap BMPO added 10–13 s a e he p epa a ion o he bu e /KO
2
solu ion
did no show any
•
BMPO-adduc spec a. The same esul was ound when KO
2
in DMSO
was added o a pH 7.4 bu e ed wa e solu ion ollowed by BMPO [6].
Using he spin ap BMPO, we obse ed ha he addi ion o a wa e solu ion o
powde KO
2
p oduced adicals, whose composi ion and in ensi y signi ican ly depended
on he pH le el du ing he dissolu ion o KO
2
. As KO
2
is a s ong base, he addi ion o a
bu e ed wa e solu ion (50 mmol L
−1
phospha e bu e , pH 7.4) o powde ed KO
2
( inal
40 mmol L−1)
inc eased he pH o 10–11. The e a e wo possible ways o dec ease he pH
o he mix u e o he physiological alue o 7.4. When pH was dec eased o 6.5–8.5 h ough
he addi ion o HCl o he p epa ed BMPO/KO
2
mix u e,
•
BMPO-OH adicals, compa ed
wi h
•
BMPO-OOH, we e mos ly obse ed a pH ~7.3–10.3 (Figu e 13). On he o he hand,
when he BMPO solu ion adjus ed wi h HCl was added o he powde ed KO
2
o ge a inal
physiological pH o ~6–8,
•
BMPO-OOH p e ailed o e
•
BMPO-OH
(Figu es 2b, 4and 10)
.
The
•
BMPO-OOH/
•
BMPO-OH a io calcula ed om he i s eco ded EPR spec um was
≥
20 (Figu e 2b). As he i s spec um was eco ded 100
±
15 s a e he BMPO–HCl
solu ion was mixed wi h powde ed KO
2
, i is e y p obable ha he
•
BMPO-OOH/OH
a io was e en highe immedia ely a e he sample p epa a ion. Based on hese da a, i
can be sugges ed ha KO
2
can be used as a sou ce o O
2•−
and
•
BMPO-OOH, when i is
ensu ed ha du ing he dissolu ion o KO
2
, he pH is wi hin he ange o 6.5–8.0. This can
be done by applying acid du ing he dissolu ion o by using a s ong physiological bu e .
Howe e , as a signi ican amoun o H
2
O
2
is p oduced du ing he dissolu ion o KO
2
by
an aqueous bu e , i s in ol emen in adical–compound in e ac ions should be also aken
in o accoun .
I is supposed ha he obse ed hyd oxyl adduc ,
•
BMPO-OH, was mos ly p o-
duced by pH- and ime-dependen decomposi ion o
•
BMPO-OOH, which was o med
by apping O
2•−
du ing KO
2
dissolu ion. This sugges ion is suppo ed by he ime- and
pH-dependen p og essi e inc ease in he ela i e concen a ion o
•
BMPO-OH, which is
mo e s able han
•
BMPO-OOH, upon a p og essi e dec ease in he
•
BMPO-OOH ela i e
concen a ion (Figu es 2b and 10). The signi ican p opo ion o he
•
BMPO-OH compo-
nen in he i s eco ded spec um a pH
≥
9.0 co ela ed well wi h published da a [
8
].
Howe e , he in ensi y o he
•
BMPO-OH spec a ansien ly inc eased o e ime in he
mix u e o BMPO + H
2
O
2
(pH 9.0 and 12.2; Figu e S1), sugges ing ha he BMPO/H
2
O
2
in e ac ion con ibu ed o he
•
BMPO-OH componen . Addi ionally, he decomposi ion o
H
2
O
2
in o eac i e oxygen species unde alkaline media may p omo e he p oduc ion o
•
BMPO-OH [
24
–
27
]. As he p esen s udy was ocused mainly on he p ac ical p epa a ion
o
•
BMPO-OOH, he mechanism o hyd oxyl adduc o ma ion was no s udied in mo e
de ail.
Ou p e ious s udy desc ibed he p epa a ion o he
•
BMPO-OOH spin adduc as a
model o he s udy o o ganic hyd ope oxide decomposi ion [
6
]. We used 10% DMSO ( / )
o p epa e he
•
BMPO adduc , which is use ul o e alua ing he an ioxidan po ency and
e ec s o compounds insoluble in wa e on
•
BMPO-OOH. In he p esen s udy,
•
BMPO-
OOH, as a po en ial model o hyd ope oxide, was p epa ed wi hou DMSO, and he
an ioxidan po ency o compounds soluble in wa e , such as H
2
S, RSe-1, and RSe-2, was
s udied. As in ou p e ious s udy wi h H
2
S in a 10% DMSO sol en [
6
,
22
], he wa e
An ioxidan s 2021,10, 1286 20 o 22
solu ion o H
2
S changed he
•
BMPO-OOH/
•
BMPO-OH p opo ion and dec eased he
o e all adical concen a ions. RSe-1 and RSe-2 in e ac ed wi h
•
BMPO adduc s and
inc eased he adical p opo ion o
•
BMPO-OH o e
•
BMPO-OOH in a ime-dependen
manne , e en in he case when he o al ime-dependen
•
BMPO adduc s we e simila o
he con ols. The de ailed mechanism o he chemical in e ac ions leading o changes in
he concen a ion and/o a io o
•
BMPO-OOH/OH is no known a p esen and equi es
u he examina ion.
The oxida ion o DNA occu s h ough eac ions wi h eac i e oxygen species (ROS),
e.g., HO
•
, O
2•−
, single oxygen (
1
O
2
), pe oxyni i e (ONOO
−
), o H
2
O
2
, which a e p o-
duced du ing endogenous biological p ocesses [
28
–
30
]. The e o e, pDNA was used o es
he biological e ec s o
•
BMPO adduc s. pDNA was signi ican ly clea ed by low
•
BMPO-
OOH/
•
BMPO-OH concen a ions, in which
•
BMPO-OOH p e ailed o e
•
BMPO-OH.
BMPO, KO
2
, H
2
O
2
, and KO
2
+ H
2
O
2
alone did no clea e pDNA, indica ing ha hey we e
no in ol ed in he pDNA clea age e ec s o
•
BMPO-OOH/
•
BMPO-OH. The mix u e
p epa ed o 10 s, which delayed he addi ion o BMPO o he bu e + HCl + KO
2
solu ion,
which did no o m a
•
BMPO adduc spec al signal, also did no clea e pDNA, con i ming
ha nei he he componen s o he mix u e no hei non adical p oduc s we e able o
clea e pDNA. Al oge he , his implies ha
•
BMPO-OOH/OH adicals a e esponsible o
pDNA clea age. A de ailed cascade o he chemical in e ac ions leading o pDNA clea age
by •BMPO-OOH/OH adicals is no known a he p esen and emains o be elucida ed.
5. Conclusions
This s udy u ilized KO
2
as a sou ce o O
2•−
adical anions and he EPR spin apping
echnique wi h BMPO o moni o he gene a ion o he
•
BMPO-OOH spin adduc and
i s con e sion o
•
BMPO-OH in a wa e solu ion wi hou DMSO as a po en ial model
o o ganic hyd ope oxides and alcohols. The concen a ion and ela i e abundance o
•
BMPO-OOH and
•
BMPO-OH elucida ed om he EPR spec a s ongly depended on
he p ocedu e o he sample p epa a ion and he pH alue. In he pH ange o 6.5–8.0,
•
BMPO-OOH p edomina ed o e
•
BMPO-OH. Mo eo e , he me hod also dis inguished
he ime-dependen concen a ion changes o bo h
•
BMPO adduc s in he same sample, so
i is app op ia e o s udies o ime-dependen in e ac ions o compounds wi h adicals.
The
•
BMPO-OOH/OH adicals clea ed he plasmid DNA. H
2
S and selenium-con aining
de i a i es inc eased he p opo ion o
•
BMPO-OH o e
•
BMPO-OOH adicals. This
demons a es ha he p esen ed app oach can be used o s udy he biological e ec s o
•BMPO-OOH/OH adicals and he in e ac ions o compounds wi h •BMPO-OOH/OH.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/
10.3390/an iox10081286/s1, P ocedu es o p epa a ion o EPR samples. Figu e S1: Time- and pH-
dependen EPR spec a o
•
BMPO adduc s o he 30 mmol L
−1
BMPO in he p esence o 1 and
10 mmol L−1H2O2.
Figu e S2: Rep esen a i e gels indica ing he e ec s o BMPO
−
KO
2
in e ac ion
on pDNA clea age a a ious pH.
Au ho Con ibu ions: Concep ualiza ion, K.O.; o mal analysis, A.M., V.B. and K.L.; in es iga ion,
A.M., V.B., M.C., K.L., M.G. and K.O.; me hodology, A.M., V.B., M.C. and K.O.; p ojec adminis a ion,
M.C. and K.O.; esou ces, K.L., M.J.N. and C.J.; supe ision, K.O.; alida ion, V.B., M.C., M.J.N., M.G.,
L.T. and K.O.; isualiza ion, A.M., V.B., M.C., M.G., L.T. and K.O.; w i ing—o iginal d a , K.O. All
au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Slo ak Resea ch and De elopmen Agency, g an numbe
APVV-19-0154 o K.O. and APVV-17-0384 o M.C., and he Scien i ic G an Agency o he Slo ak
Republic, g an numbe VEGA 1/0064/21 o V.B., 2/0079/19 o M.G., 2/0091/21 o A.M., and
2/0053/19 o M.C. V.B. hanks Minis y o Educa ion, Science, Resea ch and Spo o he Slo ak
Republic o unding wi hin he scheme “Excellen esea ch eams”.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
An ioxidan s 2021,10, 1286 21 o 22
Da a A ailabili y S a emen :
All indings and conclusions a e based on he p esen ed igu es in he
main ex o in he Supplemen a y Ma e ials. O iginal sou ce iles can be sen om he co esponding
au ho , D . Ka ol Ond ias, upon eques .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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