E ec s o Cha ged Su ace on Elec ochemical Sensi i i y o P o ein
Dime iza ion
Hana Ce nocká, Ve onika Kasalo á, E a Tihla íko á, Vilém Nedela, Roman H s ka,
and Ve onika Os a ná*
Ci e This: Anal. Chem. 2025, 97, 24787−24794
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ABSTRACT: P o ein dime iza ion is a c ucial biological p ocess in which
p o eins in e ac in o homo- o he e odime s o o m a unc ional assembly.
Unde s anding and modula ing he molecula mechanisms o p o ein
dime iza ion and hei unc ion ep esen he cu ing edge o esea ch and
p o ide mul iple en ies o biomedical applica ions. Label- ee me hods
sensi i e o homodime o ma ion a e s ill equi ed. Elec ochemical me hods
a e sensi i e o small s uc u al changes due o he p esence o a su ace and
pola iza ion o high nega i e/posi i e po en ials, whe e pa ial dena u a ion/
un olding can appea . Since he dime ic s uc u e is usually mo e lexible, he
elec ic ield e ec s induce mo e salien s uc u al changes close o he elec ode su ace, accompanied by highe
ch onopo en iome ic/ ol amme ic esponses. Se um albumin and an e io g adien ecep o -2 we e s udied as model homodime ic
p o eins.
■INTRODUCTION
P o ein dime iza ion is a c ucial biological p ocess whe e
p o eins in e ac o o m homo- o he e odime s, c ea ing
unc ional assemblies. This sel -assembly in o dime s o highe -
o de oligome ic agg ega es is a common biophysical
phenomenon occu ing in e e y cellula compa men . P o ein
dime iza ion signi ican ly egula es a ious cellula pa hways,
including enzyma ic ac i a ion, signal ansduc ion, and
pa hogenic pa hways.
1
Regula ing p o ein dime iza ion is
essen ial o he g ow h and de elopmen o o ganisms,
in luenced by in insic o ex insic ac o s in he na u al
en i onmen .
2
App oxima ely 50% o oligome ic p o eins exis
as homodime s.
3
Dime ic p o ein o ms o e po en ial
ad an ages, such as gene ic sa ings, unc ional gains, and
s uc u al bene i s in compa ison o monome ic ones. No ably,
se e al oncogenic signaling pa hways a e media ed by dime ic
p o eins, leading o cell p oli e a ion. Many o hese he e o- o
homodime ic p o eins a e key componen s in oncogenic
signaling pa hways and ha e become popula a ge s o
de eloping an i umo agen s.
4
The e o e, unde s anding and
modula ing he molecula mechanisms o p o ein dime iza ion
and hei unc ions could be use ul in pha macology as well as
o he biomedical applica ions.
The e a e many s anda d me hods based on label- ee and
label-based app oaches, which a e used o he analysis o
oligome s, including dime s. Mos o he label-based me hods
u ilize luo escen ags in a ious modes o luo escence
analysis, such as s eady-s a e luo escence, luo escence
aniso opy, Fo s e Resonance Ene gy T ans e (FRET),
e c.
5−7
Gel elec opho esis, size exclusion ch oma og aphy,
ul acen i uga ion, dynamic ligh sca e ing, nuclea magne ic
esonance, X- ay c ys allog aphy, mic oscopy, p o ein cha ge
ans e spec a, and o he s a e o en used label- ee
me hods
8−10
o his pu pose. Some o hem a e able o
dis inguish monome s and dime s based on size, and o he s
desc ibe p o ein s uc u e in de ail. Compu e simula ions o e
ex ao dina y insigh s in o in e ac ion mechanisms. I can
e lec binding con o ma ion, in e ac ion o ces, binding
a ini y, key esidues, and o he in o ma ion ha physicochem-
ical expe imen s canno e eal in a as and de ailed manne .
11
Howe e , he esul s o simula ions mus be igo ously es ed
by using physicochemical expe imen s. Elec ochemical
me hods can be used o his pu pose as echniques o
conduc ing as p elimina y es s o p o ein s uc u al changes
and o elucida ing p o ein in e ac ions wi h in e ac ing
pa ne s, including DNA, p o eins, and pep ides.
12,13
Valuable
app oaches o analyzing biological molecules a a cha ged
su ace include ol amme ic and impedance spec oscopic
me hods.
14
Cons an cu en ch onopo en iome ic s ipping
(CPS) is ano he use ul elec ochemical me hod, enabling he
acquisi ion o s uc u al and s abili y da a and p o iding
Recei ed: Augus 27, 2025
Re ised: Oc obe 21, 2025
Accep ed: Oc obe 22, 2025
Published: Oc obe 31, 2025
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addi ional insigh s in o he di e en ial dynamic beha io o he
p o eins. Like o he elec ochemical me hods, i is cha ac-
e ized by low equipmen cos s, low p ice, and ela i ely simple
ope a ion.
12
The CPS in combina ion wi h a ca aly ic
hyd ogen e olu ion eac ion allows he s udy o any known
p o eins a cha ged su aces. The cha ged su ace plays a
c ucial ole in his ype o analysis since he p o eins a e
accumula ed a he uncha ged su ace
15
whe e hey e ain hei
olded s uc u es. Subsequen ly, elec ode pola iza ion o
nega i e po en ial can lead o s uc u al change in an ex eme
case, o dena u a ion/un olding.
16
P o ein s uc u al changes
occu on a wide ange o ime scales, om he ex emely as
( em oseconds) o he ela i ely slow (mic oseconds o e en
milliseconds).
17
The exposu e ime in CPS can be limi ed o
milliseconds and con olled by he alue o he s ipping
cu en .
13
Small s uc u al changes in p o eins can a ec hei
s abili y a nega i ely cha ged in e aces, po en ially leading o
un olding. This is e lec ed in signi ican changes in CPS peak
H heigh and shape due o di e en accessibili y o
elec oac i e g oups.
18
CPS has been success ully used o
moni o a ious p o ein s uc u al changes, such as one amino
acid exchange,
19
oligome iza ion, and agg ega ion,
12,18
wi h
esul s aligning well wi h o he me hods like luo escence
spec oscopy, dynamic ligh sca e ing, gel elec opho esis, and
H/D exchange mass spec ome y.
20−22
Se e al s udies we e
done using model p o ein se um albumin
23−27
ollowed by
applica ion o biomedically impo an p o eins.
12,13
Se um albumin, wi h a molecula weigh o 66.5 kDa, he
mos abundan p o ein in human blood plasma,
28
makes up
abou hal o he se um p o ein a concen a ions be ween 526
and 753 μM.
29
Besides main aining plasma onco ic p essu e,
se um albumin has a ious unc ions, such as anspo ing
s e oids and binding o eac i e oxygen species.
6
The human
se um albumin (HSA) dime s se e as bioma ke s o oxida i e
s ess and li e ci hosis.
30
Unde physiological condi ions,
albumin can unde go concen a ion-dependen , e e sible sel -
oligome iza ion,
10
accompanied by a con o ma ional change
om a hea -shaped e ia y s uc u e o an ellipsoid in
solu ion.
31
In his wo k, we compa ed CPS esponses o monome s and
dime s/oligome s, and we show ha CPS can dis inguish
be ween hem u ilizing he di e en adso p ion o hese
indi idual o ms on a cha ged su ace. CPS peak heigh o
na i e un ea ed bo ine se um albumin (BSA) is be ween he
peaks o he monome ic and dime ic o ms. Simila esul s
we e obse ed by using ol amme y a ca bon elec odes.
Besides se um albumin, we we e able o ollow edox-
dependen dime iza ion o he p o ein an e io g adien
ecep o 2 (AGR2).
■MATERIALS AND METHODS
Ma e ials. All chemicals and eagen s we e pu chased om
Me ck (Czech Republic) o he highes a ailable quali y,
mos ly o analy ical g ade. Solu ions we e p epa ed om iple-
dis illed wa e .
Bo ine se um albumin (BSA, M.W. 66 432 Da) was
ob ained om Me ck (Czech Republic).
AGR221−175 (lacking signal pep ide) cloned in o pEHISTEV
was kindly p o ided by P o . T. Hupp.
32
AGR2 p o ein was
p epa ed using a QuikChange Si e-Di ec ed Mu agenesis Ki
(S a agene) acco ding o he manu ac u e ’s ins uc ions.
Pu i ica ion o AGR2 p o ein was desc ibed ea lie .
21,33
Pu i ied usion His6-AGR2 p o ein was subsequen ly clea ed
wi h he His6-TEV p o ease o emo e he His6- ag. The N-
e minal His6- ag o AGR2 wi h His6-TEV p o ease was hen
cap u ed using a HisT ap FF 5 mL column (GE Heal hca e),
whe eas he pu i ied ecombinan p o ein was p esen in he
low- h ough ac ions. A e pu i ica ion, he p o ein concen-
a ion was de e mined spec opho ome ically by using he
mola ex inc ion coe icien ob ained om he P o Pa am
so wa e on he EXPASY se e .
P ocedu es. Dena u a ion. BSA a 100 μM concen a ion
in iply dis illed wa e was ea ed a 95 °C o 30 min,
ollowed by cooling o oom empe a u e in a wa e ba h.
Monome s Dime /Oligome Sepa a ion Using Fil a-
ion. BSA solu ion was ea ed h ough a Mic ocon 100k
memb ane il e (Me ck, Ge many) wi h a 100 kDa cu o by 5
min il e ing a 13400 pm o sepa a e monome s and highe
oligome s.
T ea men wi h Hyd ogen Pe oxide. A 20 μM amoun o
AGR2 was incuba ed wi h di e en H2O2concen a ions
o e nigh .
Elec ochemical Analysis. Ch onopo en iome ic S ip-
ping. Ch onopo en iome ic s ipping was pe o med using an
Au olab analyze (PGSTAT302, Me ohm-EcoChemie, The
Ne he lands) connec ed wi h VA-S and 663 (Me ohm,
Swi ze land). A h ee-elec ode sys em in a s anda d he mo-
s a ic cell was used: hanging me cu y d op elec ode (HMDE,
0.4 mm2) as he wo king elec ode, Ag|AgCl|3 M KCl as he
e e ence, and P wi e as he auxilia y elec ode. Da a was
collec ed using GPES e sion 4.9.007. Expe imen s we e
eplica ed a leas 3 imes o each measu emen . The ela i e
s anda d de ia ion (RSD) o ol amme ic measu emen s a
he HMDE did no exceed 6%. Fo mos expe imen s,
adso p i e s ipping ans e was pe o med: 1 μM BSA in
50 mM Na-phospha e, pH 7.0, was adso bed a he HMDE
om a 5 μL d op a an open cu en ci cui o he
accumula ion ime Ao 60 s and ans e ed o he backg ound
elec oly e, 50 mM Na-phospha e, pH 7.0, whe e he
ch onopo en iog am was eco ded wi h a s ipping cu en ,
Is ,o −70 μA a e a 5 s exposu e a a po en ial o EBo −0.3
V (i no s a ed o he wise). The ini ial po en ial was he same
as ha o EB. Measu emen s we e acqui ed in open ai a 25 °C
o se um albumin and a 18 °C o AGR2 p o ein.
Squa e Wa e Vol amme y. An edge plane py oly ic
g aphi e elec ode (ePGE, 4 mm2) o a glassy ca bon elec ode
(GCE, 3.14 mm2) was used as he wo king elec ode. The
samples we e adso bed a he ca bon elec ode om 8 μL
d ops a an open ci cui po en ial o a Ao 5 min. The
elec ode was washed wi h wa e and ans e ed o he
backg ound elec oly e in he elec ochemical cell. The
ol ammog ams we e measu ed om 0.4 o 1.3 V a an
ampli ude o 10 mV, a s ep o 10 mV, and a equency o 26 Hz
unde a gon.
AGR2 expe imen s we e pe o med in 0.1 M Na-phospha e,
pH 7.0, using GCE a 18 °C, while hose wi h BSA we e
pe o med in 50 mM Na-phospha e, pH 7.0, a ePGE and 25
°C.
Di e en ial Scanning Calo ime y. The mog ams o 47
μM BSA dena u a ion in 50 mM Na-phospha e, pH 7.0, we e
eco ded using a NanoDSC di e en ial scanning calo ime e
(TA Ins umen s, New Cas le, USA). The degassed p o ein
solu ion was hea ed om 293 o 363 K a a cons an a e o 1
K·min−1in a 300 μL capilla y cell o he calo ime e . The
e e ence cell was illed wi h 50 mM Na-phospha e, pH 7.0.
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Elec opho e ic Mobili y Shi Assay. Na i e PAGE was
pe o med on No ex WedgeWell T is-Glycine Mini P o ein
Gel, 4−20% (The moFishe Scien i ic). Fi e μg o BSA was
loaded on he gel, and PAGE was un in 25 mM T is and 192
mM glycine unning bu e a 225 V o 1 h. The gel was
s ained in a Coomassie B illian Blue solu ion.
Fluo escence Spec oscopy. Fluo escence spec a o 2
and 20 μM BSA in 50 mM Na-phospha e, pH 7.0, we e
measu ed a an exci a ion wa eleng h o 280 nm wi h an ISS
PC1 pho on coun ing spec o luo ome e (ISS, USA) in a
qua z cu e e wi h a 1 cm pa h leng h. The exci a ion and
emission sli wid hs we e ixed a 4 nm each.
Ci cula Dich oism Spec ome y. Ci cula dich oism
spec a o 2 μM BSA in 10 mM Na-phospha e, pH 7.0, we e
measu ed om 180 o 300 nm a 100 nm·min−1scan speed in
a qua z cu e e wi h a 1 mm pa h leng h unde ni ogen as he
a e age o ou accumula ions. A J-815 dich og aph (Jasco,
Japan) was used o he measu emen s.
Elec on Mic oscopy. Monome ic, dime ic, and oligo-
me ic BSA we e s udied using an en i onmen al scanning
elec on mic oscope (ESEM) Quan a 650 FEG, which is well-
sui ed o he obse a ion o a wide ange o samples.
34,35
P esen ed images we e ob ained in da k ield (DF) mode wi h
a de ec o o scanning ansmission elec on mic oscopy
(STEM). Obse a ion was ealized in high acuum mode o
he ESEM
36
using elec on beam ene gy o 30 keV and cu en
o 20 pA. Samples we e p epa ed by applica ion o 1 μL o 15
μM BSA solu ions on a TEM g id co e ed wi h a holey ca bon
ilm and ai -d ied.
■RESULTS AND DISCUSSION
Ch onopo en iome ic S ipping o Monome ic and
Dime ic/Oligome ic Se um Albumin. We analyzed BSA
monome s and dime s using CPS and o he me hods.
Monome s and dime s/oligome s we e p epa ed by il a ion
h ough a Mic ocon 100 kDa. The monome s wi h a MW o
66 kDa passed h ough he il e , while dime s and highe
oligome s s ayed a he uppe pa o he il e . Such p epa ed
samples we e cha ac e ized by he na i e mobili y shi assay
and used o u he analyses. Monome s ha passed h ough
he il e yielded a s onge band a MW 66 kDa (Figu e 1A,
inse ), and dime s/oligome s yielded bands co esponding o
di e en qua e na y s uc u es (monome s, dime s, and highe
oligome s in lowe amoun s). The p esence o monome s
could be due o he disagg ega ion o nonco alen dime s/
oligome s by he elec ic ield e ec s du ing elec opho esis.
37
In CPS, 1 μM BSA was adso bed om a 5 μL d op on
HMDE o an accumula ion ime, A, o 60 s a open ci cui .
Then, he BSA-modi ied elec ode was exposed o he
po en ial, EB, usually o −0.3 V o 5 s, accompanied by
s i ing, ollowed by CPS peak H eco ding. Bo h samples,
monome ic and dime ic/oligome ic BSA, p oduced a small
peak H (abou 0.4 s/V a Is −70 μA) a po en ial −1.85 V
(Figu e 1A) in compa ison o he mally dena u ed BSA,
eaching 2.5 s/V. The peak H o monome ic BSA was abou
30% smalle han ha o he dime ic/oligome ic BSA o m.
Acco ding o he li e a u e, dime o ma ion leads o a mo e
hyd ophobic en i onmen o he ligand-binding pocke .
6
A
mo e hyd ophobic en i onmen p omo es s onge adso p ion
a he me cu y su ace, esul ing in a highe peak H. A simila
end was obse ed o ano he p o ein, galec in-1. In ou
s udy,
38
a smalle peak H was eco ded o monome ic
galec in-1 incuba ed a 2 μM, whe eas a signi ican ly highe
peak H was de ec ed o he homodime ic o m incuba ed a
14 μM, sugges ing a shi owa d dime o ma ion a highe
concen a ions. The dime iza ion cons an o galec in-1 was
de e mined o be a ound 5 μM.
39
These esul s suppo he
no ion ha dime iza ion enhances galec in-1 in e ac ion wi h
he elec ode su ace, p obably due o inc eased hyd o-
phobici y combined wi h educed s uc u al compac ness.
The di e ence be ween monome s and dime s/oligome s
was obse ed o a ious su ace concen a ions, changing
acco ding o he accumula ion ime, A(Figu e 1B). The
highes peak H heigh s o monome s a e achie ed a sho e A
(abou 90 s) han o dime s/oligome s ( A> 200 s) p obably
due o he di e en di usion coe icien s o monome s and
dime s/oligome s and/o p e e en ial adso p ion o monome s
on he elec ode su ace. Di e ences in peak H heigh e lec
he accessibili y o elec oac i e species.
12,18
Mo e amino acid
esidues, esponsible o highe peak H, a e accessible in
dime s/oligome s han in monome s,
18
which could be a esul
o he di e en way o adso p ion. The adso p ion o bo h
species could be in luenced no only by di e ences be ween
monome s and dime s/oligome s hemsel es, bu also due o
con o ma ional changes o he monome ic subuni s in dime s/
oligome s in compa ison o he monome s uc u e.
40
The dependence o he peak H heigh o bo h o ms on
po en ial EBdi e ed (Figu e 1C). A posi i ely cha ged and
sligh ly nega i ely cha ged elec ode, he peak heigh a io o
bo h o ms o BSA changed only sligh ly. The exposu e o
po en ial EB om −1.0 V o −1.5 V induced a g adual inc ease
in he di e ence be ween monome ic and dime ic/oligome ic
Figu e 1. (A) CPS peaks H o 1 μM monome ic (black), dime ic/oligome ic ( ed), and he mally dena u ed BSA (blue) in backg ound elec oly e,
e. Inse : Na i e PAGE o BSA was pe o med o wo ac ions a e il a ion h ough a Mic ocon. (B,C) Dependence o CPS peak heigh o
monome ic (black) and dime ic/oligome ic BSA ( ed) on (B) accumula ion ime, A, and (C) po en ial, EB, applied be o e ch onopo en iog am
eco ding o 5 s. Is =−70 μA.
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o ms, wi h he highes di e ence a −1.5 V, whe e he
dime ic/oligome ic o m yielded peak H wice as high as he
monome ic o m. A highly nega i e po en ials, mo e han
−1.55 V, bo h samples we e deso bed om he su ace,
accompanied by a peak H dec ease. The da a o he EB
dependence show ha applying he po en ial, EB, a he
p o ein-modi ied elec ode can enhance di e ences be ween
monome s and dime s/oligome s.
S abili y o Monome s and Dime s. The CPS peak H
heigh o he dime ic/oligome ic o m a po en ial −1.5 V was
mo e han wice as high as a a po en ial o −0.3 V, while he
peak H o he monome ic o m inc eased by less han 40%
(Figu e 1C), which sugges ed i s highe s abili y agains
elec ic ield e ec s. A simila conclusion can be d awn om
he dependence o CPS peak H on s ipping cu en , Is
(Figu e 2A), which shows a shi o Is 1/2 alues o highe
nega i e alues o he dime ic o m.
18
This shi implies ha
sho e exposu e o nega i e po en ial induces s uc u al
p o ein change.
13,16
Dime ic/oligome ic o ms showed lowe
s abili y no only agains he elec ic ield e ec (Figu es 1C
and 2A), bu also agains he mal dena u a ion, as di e en ial
scanning calo ime y p o ed (Figu e 2B). 47 μM BSA in 50
mM phospha e bu e , pH 7.0, in a inal olume o 300 μL was
hea ed om 20 o 90 °C wi h a hea ing a e o 1 °C/min. The
dime /oligome s dena u a ion begins a a Tio abou 42.4 °C
and eaches he dena u a ion empe a u e (Td) o abou 59.2
°C, while o monome , a Tio abou 46.4 °C and Tdo 59.9
°C we e obse ed, in ag eemen wi h da a.
41
The en halpy o
dena u a ion, ΔH, was abou 517.5 kJ mol−1 o dime s/
oligome s and abou 687.5 kJ mol−1 o monome ic BSA,
showing he highe he mal s abili y o monome s. The
luo escence spec a a he wo concen a ions di e ed only
in he ange o expe imen al e o s (Figu e 2C,D). One
concen a ion was chosen, simila o ha a which elec o-
chemical expe imen s we e pe o med, and he second was en
imes highe . We es ed wo concen a ions because esea ch
by Bha acha ya e al. wi h na i e BSA and HSA shows hei
di e en beha io a lowe and highe concen a ions han 10
μM.
9
The au ho s explained his as a esul o he dime iza ion.
Howe e , we also obse ed se e al imes highe luo escence
Figu e 2. Monome s’ (black) and dime s/oligome s’ ( ed) s abili y. (A) Dependence o CPS peak heigh on s ipping cu en o 1 μM BSA o ms.
(B) Di e en ial scanning calo ime y o 47 μM BSA monome s and dime s/oligome s. (C, D) Fluo escence spec a and (E) ci cula dich oism o
(C, E) 2 μM and (D) 20 μM BSA o ms. (F−H) STEM images o (F) monome ic, (G) dime ic/oligome ic, and (H) highe oligome ic BSA laye .
Highe MW oligome s we e p epa ed by ea men a 90 °C o 1 min.
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spec a o 20 μM BSA han o 2 μM, bu no di e ence
be ween monome s and dime s/oligome s (Figu e 2C,D).
Spec a a bo h concen a ions sugges he same en i onmen
o y osine and yp ophan esidues in monome ic and
dime ic/oligome ic o ms in solu ions. Likewise, he di e -
ences obse ed using ci cula dich oism we e negligible
(Figu e 2E), sugges ing only mino s uc u al changes in he
albumin s uc u e o monome s and dime s/oligome s. The
au ho s in e 9assumed he o ma ion dime by changing
albumin concen a ions. The changes in luo escence and
ci cula dich oism spec a ob ained in he men ioned
publica ion a e p obably due o phenomena o he han
dime iza ion.
Insigni ican changes be ween laye s o monome s and
dime s we e also p o ed by STEM isualiza ion in he da k-
ield mode.
18
Monome ic (Figu e 2F) and dime ic BSA
(Figu e 2G), as small b igh spo s (app oxima ely up o 50 nm
in diame e ), a e e enly dis ibu ed. Bigge amoun s and mo e
in ense b igh smea s we e obse ed o oligome s ecei ed
a e 1 min o incuba ion a 90 °C (Figu e 2H).
Signi ican di e ences in elec ochemical esponses and
negligible ones in luo escen spec a, ci cula dich oism, and
STEM images could be due o he p esence o a cha ged
su ace. The su ace i sel may enhance di e ences be ween
monome s and dime s and oligome s by hei di e en
adso p ion. Su ace pola iza ion can u he inc ease he
di e ences in CPS esponses o monome s and dime s/
oligome s due o hei a ious des abiliza ion by elec ic ield
e ec s. The elec ic ield e ec s a e a esul o he combina ion
o he applied po en ial and exposu e ime. A p ope
combina ion o applied po en ial and exposu e ime is
impo an o he equi ed di e ences in esponses be ween
monome s and dime s/oligome s. Fo ins ance, a Is alue o
−100 μA, he peak H heigh s o bo h o ms a e almos he
same, since he exposu e ime is e y sho (abou 1 ms).
Concen a ion Dependence. Va ious sensi i i ies o
changes be ween monome s and homodime s o di e en
me hods could be dependen on he p o ein concen a ions
equi ed o he analyses. The esul s om di e en me hods
could be in luenced by he p o ein oligome iza ion s a us since
concen a ion is one o i s egula o y ac o s.
10
The e ia y and
qua e na y s uc u e o HSA depends on he o e all HSA
concen a ion in solu ion and na u ally al e s he p o ein
beha io .
6
We pe o med CPS analysis o bo h o ms
depending on he concen a ion. Since he CPS peak H
heigh /a ea depends on concen a ion, we incuba ed se um
albumin samples a di e en concen a ions o e nigh , and
hen he incuba ed samples we e dilu ed o 1 μM
concen a ion immedia ely be o e analysis.
Figu e 3. Dependence o peak H heigh on concen a ion o (A) monome s, (B) dime s/oligome s, and (C) un ea ed na i e BSA a Is =−60 μA.
The dashed lines ep esen a e age alues o monome s (black), dime s/oligome s ( ed), and un ea ed BSA ( iole ), whe e he a e age alue is
calcula ed o each concen a ion.
Figu e 4. Baseline-co ec ed squa e wa e ol amme ic YW peak a glassy ca bon elec ode. (A) One μM monome ic (black), dime ic/oligome ic
( ed), un ea ed (pu ple), and dena u ed BSA (blue) a edge-o ien ed py oly ic g aphi e elec ode. (B) Fi e μM na i e (o ange) and oxidized
AGR2 (g een) by 125 μM hyd ogen pe oxide. (C) Dependence o peak YW heigh on incuba ion wi h di e en concen a ions o hyd ogen
pe oxide analyzed in 0.1 M Na-phospha e, pH 7.0. Inse : SDS PAGE o AGR2 incuba ed wi h a ious hyd ogen pe oxide concen a ions. Adap ed
wi h pe mission om e 47. Copy igh 2016 Else ie . (D) CPS peak H o 2 μM AGR2, Is =−15 μA. Inse : SDS PAGE o AGR2 in he absence
and p esence o educing agen DTT. Adap ed wi h pe mission om e 47. Copy igh 2016 Else ie .
Analy ical Chemis y pubs.acs.o g/ac A icle
h ps://doi.o g/10.1021/acs.analchem.5c05281
Anal. Chem. 2025, 97, 24787−24794
24791
Peak H heigh o monome ic BSA incuba ed o e nigh
eached alues be ween 0.35 and 0.64 s/V (Figu e 3A), while
dime ic BSA eached alues be ween 0.75 and 1.62 s/V
(Figu e 3B) a Is =−60 μA. The a e age peak H heigh o
un ea ed BSA a concen a ions o 1 and 2 μM was abou 0.75
s/V. This alue be ween hose o monome s and dime s
(Figu e 3C) sugges s he p esence o monome s and dime s/
oligome s in he un ea ed BSA sample. We ob ained only an
insigni ican inc ease o peak H heigh s a highe concen-
a ions han 10 μM, which is a concen a ion ha should ha e
caused dime o ma ion as desc ibed Lahi i e al.
6
Howe e ,
he equilib ium be ween monome s and dime s/oligome s can
be shi ed on he elec ode in compa ison o solu ion.
40
A e
o e nigh incuba ion, he di e ence be ween he wo o ms
s ill emained (Figu e 3A,B). This ac con adic s he
obse a ion o Bha acha ya e al.
9
desc ibing as associa ion
and dissocia ion o monome s o dime s and ice e sa
depending on concen a ion. The disag eemen s be ween ou
esul s and hose in e 9can be due o mo e complex
p ocesses in ol ed in p o ein concen a ion inc ease, no jus
simple associa ion wi h dime s. One can be, o ins ance, a
o ma ion o co alen and nonco alen dime s. Each
expe imen al de ail can play an impo an ole.
Monome s and Dime s/Oligome s a Ca bon Elec o-
des. We we e in e es ed in whe he he di e ence be ween
monome ic and oligome ic o ms is due o he cha ged su ace
and/o he special p ope ies o he me cu y elec ode su ace.
Besides he me cu y elec ode, he s uc u e-sensi i e analysis
can be pe o med a g aphi e elec odes,
12
whe e he bes
esul s o na i e and dena u ed se um albumin we e obse ed
a an edge-o ien ed py oly ic g aphi e elec ode and a glassy
ca bon elec ode.
42
In con as o he me cu y elec ode
pola ized o nega i e po en ials, ca bon-based elec odes a e
pola ized o posi i e po en ials.
13,43
Un ea ed na i e, mono-
me ic, and dime ic/oligome ic BSA a 1 μM concen a ion
we e adso bed a ePGE o 5 min, and hen he modi ied
elec ode was washed and ans e ed o an elec oanaly ical
cell, whe e squa e-wa e ol amme ic analysis was pe o med.
Likewise, a he HMDE, we obse ed di e ences be ween
un ea ed, monome ic, and dime ic/oligome ic o ms, whe e
monome ic and un ea ed BSA yielded oxida ion peaks o
y osine and yp ophan (peak YW) abou 7 imes smalle han
ha o he mally dena u ed BSA
42
(Figu e 4A).
Dime iza ion o se um albumin in plasma has majo
implica ions o no mal physiology, since he p esence o
albumin dime s educes he osmo ic p essu e.
44
Apa om
se um albumin, plen y o p o eins a e ac i e in hei
homodime ic o m. We es ed an e io g adien p o ein 2
(AGR2), which was well cha ac e ized by CPS analysis
alone,
21,33
and also in he e odime ic complexes.
45
This p o ein
is an impo an dime ic p o-oncogenic p o ein in ol ed also in
espi a o y and diges i e diseases.
46
Cla ke e al. p o ed using
biochemical assays and elec osp ay ioniza ion mass spec om-
e y
47
ha low le els o a chemical oxidan p omo e an
in e molecula disul ide bond h ough he o ma ion o a labile
sul enic acid in e media e. Howe e , highe le els o he
oxidan p omo e sul inic o sul onic acid o ma ion, hus
p e en ing co alen dime iza ion o AGR2. The edox-
dependen monome ic-dime ic o ma ion has implica ions o
he edox egula ion o he p o-oncogenic unc ions o AGR2
p o ein in cance cells.
48,49
We es ed whe he elec ochemical
analysis is sensi i e o edox-dependen dime iza ion o he
AGR2 p o ein (Figu e 4B−D). Wild ype (w ) AGR2 was
incuba ed o e nigh wi h di e en concen a ions o hyd ogen
pe oxide, ollowed by ol ame ic analysis a a glassy ca bon
elec ode. A signi ican ly highe peak YW was obse ed o
AGR2 ea ed wi h H2O2, be ween 25 and 175 μM (Figu e
4C), which is in good ag eemen wi h da a ob ained using he
PAGE mobili y shi assay published by Cla ke e al.
47
Addi ionally, he CPS peak H heigh o na i e AGR2 and
ha oxidized by 125 μM H2O2co espond well o he
monome ic and dime ic o ms unde he edox condi ions
(Figu e 4D).
■CONCLUSION
P o ein dime iza ion is impo an in many p ocesses in he cell
and ep esen s a key s ep in he espec i e signaling cascade.
Dime s as unc ional assemblies a e less igid han monome s,
al hough he changes in unc ionali y do no equi e la ge
con o ma ional changes.
3
In his wo k we show ha elec o-
chemical me hods can moni o p ocesses linked o dime iza-
ion. The s uc u e o he monome and i s analogue in dime s
di e s only negligibly; he e o e, s udying di e ences be ween
hem is no i ial. Mos o he label- ee me hods ha s udy
p o eins in solu ion do no see he di e ence be ween hem,
only hose ha analyze he di e ence in MW and adius, such
as elec opho e ic mobili y shi assay, dynamic ligh sca e ing,
e c. Howe e , hese me hods a e no sensi i e o small changes
in he s uc u e and unc ionali y.
The ad an age o elec ochemical me hods in p o ein
analysis is ha p o eins a e s udied a he su aces, which a e
pola ized. P o ein adso p ion on he su ace can esul in he
pa ial un olding o he pa close o he elec ode. E en small
con o ma ional changes can in luence p o ein adso p ion, in
ou case, se um albumin, a he elec ode su ace. Addi ionally,
he elec ode pola iza ion o high nega i e/posi i e po en ials
leads o u he pa ial un olding o he BSA s uc u e in pa s
close o he elec ode. Pa ial dena u a ion o he less igid
dime ic s uc u e esul ed in a highe ch onopo en iome ic
peak H as well as a highe ol ame ic peak YW. Besides BSA, a
simila end was obse ed o na i e AGR2 and i s oxidized
o m. Thus, ou esul s show he possibili y o u ilizing
elec ochemical analysis o homodime ic p o eins, which can
ha e a signi ican impac on unde s anding biochemical
p ocesses, such as in he case o galec in-1.
38
■AUTHOR INFORMATION
Co esponding Au ho
Ve onika Os a ná −Ins i u e o Biophysics, The Czech
Academy o Sciences, . .i., 61200 B no, Czech Republic;
o cid.o g/0000-0001-5721-1608; Email: [email p o ec ed]
Au ho s
Hana C
e nocká −Ins i u e o Biophysics, The Czech Academy
o Sciences, . .i., 61200 B no, Czech Republic; o cid.o g/
0000-0002-1673-1305
Ve onika Kasalo á −Ins i u e o Biophysics, The Czech
Academy o Sciences, . .i., 61200 B no, Czech Republic
E a Tihla
íko á −Ins i u e o Scien i ic Ins umen s, The
Czech Academy o Sciences, . .i., 61264 B no, Czech
Republic; o cid.o g/0000-0002-7983-2971
Vilém Nede
la −Ins i u e o Scien i ic Ins umen s, The Czech
Academy o Sciences, . .i., 61264 B no, Czech Republic;
o cid.o g/0000-0001-6029-5435
Analy ical Chemis y pubs.acs.o g/ac A icle
h ps://doi.o g/10.1021/acs.analchem.5c05281
Anal. Chem. 2025, 97, 24787−24794
24792
Roman H s ka −Masa yk Memo ial Cance Ins i u e,
Regional Cen e o Applied Molecula Oncology, 656 53
B no, Czech Republic; o cid.o g/0000-0002-6139-2664
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acs.analchem.5c05281
Au ho Con ibu ions
The manusc ip was w i en h ough he con ibu ions o all
au ho s. All au ho s ha e gi en app o al o he inal e sion o
he manusc ip .
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This esea ch was suppo ed by he p ojec No. 23−06115S o
he Czech Science Founda ion, by he p ojec SALVAGE (P
JAC; eg. no. CZ.02.01.01/00/22_008/0004644) −co unded
by he Eu opean Union and by he S a e Budge o he Czech
Republic and by MH CZ - DRO (MMCI, 00209805). The
AV21 S a egy P ojec “B eaking Technologies o he Fu u e -
Sensing, Digi isa ion, A i icial In eligence and Quan um
Technologies” unded his esea ch as well. The au ho s
hank I a Kejno ska o he assis ance wi h ci cula dich oism
expe imen s.
■ABBREVIATIONS
AGR2,an e io g adien ecep o 2; BSA,bo ine se um
albumin; CPS,cons an cu en ch onopo en iome ic s ip-
ping; DTT,di hio h ei ol; EB,applied po en ial; ePGE,edge
plane py oly ic g aphi e elec ode; GCE,glassy ca bon
elec ode; HMDE,hanging me cu y d op elec ode; HSA,hu-
man se um albumin; Is ,s ipping cu en ; A,accumula ion
ime; YW peak,oxida ion peak o y osine and yp ophan.
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