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ISOLATION OF BRANCHED CHAIN AMINOACIDS (VALINE, LEUCINE AND ARGININE)
USING MAGHEMITE PARTICLES
1,2
Na alia CERNEI,
1,2
Pa el KOPEL,
3
Pa el HORKY,
1,2
Da id HYNEK,
1,2
Ond ej ZITKA,
1,2
Voj ech ADAM
1
Depa men o Chemis y and Biochemis y, Facul y o Ag iSciences, Mendel Uni e si y in B no,
Czech Republic, EU, oj [email protected]
2
Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech Republic, EU
3
Depa men o Animal Nu i ion and Fo age P oduc ion, Facul y o Ag iSciences, Mendel Uni e si y in B no,
B no, Czech Republic, EU
Abs ac
In his pape , we epo he op imal condi ions o he isola ion and p econcen a ion o b anched chain amino
acids (BCAAs) - leucine, aline and a ginine om di e en ypes o ma ix, such as issue, plasma, blood,
bac e ia o cells. He ein, we ocused on he syn hesis o pa amagne ic pa icles able o isola e and immobilize
amino acids and hus p econcen a e i o subsequen analysis on ion-exchange liquid ch oma og aphy wi h
VIS de ec o . We modi ied nanomaghemi e (γ-Fe
2
O
3
) by mul i walled ca bon nano ubes (MWCNT) and
hyalu onic acid as he unc ional ca ie s p o iding excellen a ini y p ope ies. Ou pa amagne ic pa icles
ha e po en ial o be e isola ion o BCAAs mainly om plasma o bac e ia and in he u u e hey can also be
applied as a pla o m o deli e y sys em.
Keywo ds: Ion Exchange Ch oma og aphy, Maghemi e, MWCNT, BCAAs, aline, leucine, a ginine
1. INTRODUCTION
B anched chain amino acids (BCAAs) play me abolic and physiologic oles in he p ocesses in mammalian
bodies, o example p o ein syn hesis [1], signalling pa hways [2], and me abolism o glucose, may be used as
possible ma ke o diabe es melli us ype 2. Oxida ion o BCAAs may inc ease a y acid oxida ion and play a
ole in obesi y. BCAAs, play majo physiological oles in he b ain unc ion and/o in immuni y [3]. They a e
in ol ed, o example in he ca diac hype ophy in egula ion o p o eosyn hesis [4]. BCAAs a e used in many
cases as a bioac i e pa o ca ionic pep ides wi h an imic obial ac i i y (mos ly leucine- ich, a ginine- ich and
aline- ich, ha ing high an ibac e ial ac i i y and pe ec hemocompa ibili y) [5,6]. Pa amagne ic pa icles
(PMPs) a e e y impo an o a ge ed d ug deli e y di ec ly o he cen e o he disease unde di e en
condi ions and, he eby, ea i pu posely and/o o enhance he in e ac ions be ween deli e y sys em and he
selec ed a ge (cance cells, bac e ia’s cells e c.) [7]. Maghemi e pa icles o isola ion o p econcen a ion o
BCAAs om luids o he body may be applicable as magne ically-immobilizable ecogni ion elemen s in
biosenso s. Nowadays, a ious me hods a e de eloped o b anched chain amino acids (BCAAs) such as
aline, leucine and a ginine de e mina ion. Mos o hem a e based on ch oma og aphy (GC, LC, UHPLC) [8]
wi h andem o mass spec ome y (MS) [9], IEC [10] o CE [11]. In ou pape , we decided o use ion exchange
ch oma og aphy wi h pos -column ninhyd in de i a iza ion and VIS de ec o o aline, leucine and a ginine
de ec ion [10].
2. METHODOLOGICAL BASES AND EXPERIMENTAL PART
2.1. Chemicals
A ginine o pu i y 99 % was ob ained om Sigma Ald ich (S . Louis, Missou i, USA). Solu ion o a ginine o
p epa ing o calib a ion cu e was p epa ed in he dilu ion bu e Na: TDG (N
3
Na-0.10 g, NaCl-
h ps://doi.o g/10.37904/nanocon.2019.8497
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11.5g, C
6
H
8
O
7
-14 pe 1L H
2
O). Fo expe imen we e used Ci ic acid, NaCl,N
3
Na,TDG,HCl 35 % om Sigma
- Ald ich (S . Louis, Missou i USA), Ninhyd in om Sigma - Ald ich (S . Louis, Missou i,USA), Me hylcellosol e
(Ingos, P ague, Czech Republic), SnCl
2
(Ingos, P ague, CzechRepublic).
2.2. Ionex ch oma og aphy
An AAA 400 (Ingos, Czech Republic) liquid ch oma og aphy appa a us was used o de e mina ion o amino
acids. The sys em consis s o a glassy illing ch oma og aphic column and s eel p ecolumn, wo
ch oma og aphic pumps o anspo o elu ion bu e s and de i a iza ion eagen , cooled ca ousel o 25 es
ubes o 1.5-2.0 mL olume, dosing al e, hea eac o , VIS de ec o and cooled chambe o de i a iza ion
eagen . Ch oma og aphic columns o ans e o elu ion bu e s and de i a iza ion eagen a e able o wo k
a low 0.01-10 mL•min
−1
unde a maximum p essu e o 40 MPa. Volume o injec ed sample was 100 µL wi h
an accu acy o applica ion RSD o abou 1%. A wo-channel VIS de ec o wi h a 5 µL low olume cu e e was
ope a ed a wa eleng hs o 440 and 570 nm.
2.3. SEM cha ac e iza ion o modi ied maghemi e pa icles
S uc u e and elemen al composi ion o pa amagne ic mic opa icles we e cha ac e ised by elec on
mic oscope. Fo documen a ion o he selec ed nanoma e ials a FEG-SEM MIRA XMU (Tescan, a.s., B no,
Czech Republic) was used. This model is equipped wi h a high b igh ness Scho ky ield emi e o low noise
imaging a as scanning a es. The SEM was i ed wi h E e ha -Th onley ype o SE de ec o , high speed
YAG scin illa o based BSE de ec o , panch oma ic CL De ec o and EDX spec ome e . The MIRA 3 XMU
sys em is based on a la ge specimen chambe wi h mo o ized s age mo emen s 130×130 mm. Samples we e
coa ed by 10 nm o ca bon o p e en sample cha ging. A ca bon coa e K950X (Quo um Technologies,
G ins ead, Uni ed Kingdom) was used. Fo au oma ed acquisi ion o selec ed a eas a TESCAN p op ie a y
so wa e ool called Image Snappe was used. The Accep ed A icle so wa e enables au oma ic acquisi ion o
selec ed a eas wi h de ined esolu ion. Di e en condi ions we e op imized in o de o each ei he minimum
analysis ime o maximum de ail du ing o e nigh au oma ed analysis. An accele a ing ol age o 15 kV and
beam cu en s abou 1 nA gi es sa is ac o y esul s ega ding maximum h oughpu .
2.4. SCEM de e mina ion o ela i e cu en esponse o modi ied maghemi e pa icles PMPs
Iden i ica ion o he ela i e cu en esponse be o e and a e BCAA binding was pe o med using a scanning
elec ochemical mic oscope, Model 920D (CH ins umen s, Aus in, TX, USA). Measu emen s we e ca ied ou
wi h 10 μL o pa amagne ic a icles in concen a ion o 10 μg·mL
−1
mixed in ACS wa e . The elec ochemical
mic oscope consis ed o a 10 mm pla inum disc p obe elec ode wi h a po en ial o 0.2 V. Ano he pla inum
disc elec ode was used wi h an O- ing as he conduc ing subs a e wi h a po en ial o 0.3 V. Du ing scanning,
he pa icles we e a ached o he subs a e pla inum elec ode by magne ic o ce om a neodymium magne .
The liquid used in he analyses consis ed o 5 % e ocene in me hanol (w/ ) mixed in a io 1:1 wi h 0.05 %
KCl in wa e ( / ). Measu emen s we e pe o med in a Te lon cell wi h olume o 1.5 mL acco ding o he
ollowing pa ame e s: ampe ome ic mode, e ical scan a ea 40 μm × 40 μm, and scan a e o 30 μm·s
−1
.
2.5. X- ay luo escence analysis o PMPs
XRF elemen al analysis o PMPs was ca ied ou on Xepos (SPECTRO analy ical ins umen s GmbH, Kle e,
Ge many) i ed wi h h ee de ec o s: Ba kla sca e - aluminium oxide, Ba kla sca e - HOPG and
Comp on/seconda y molybdenum espec i ely. Analyses we e conduc ed in Tu bo Quan cu e e me hod o
measu ing. Analysis pa ame e s we e se o - measu emen du a ion: 300 s, ube ol age om 24.81 o 47.72
kV, ube cu en om 0.55 o 1.0 mA, wi h ze o peak a 5000 cps and acuum swi ched o .
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2.6. P epa a ion o mic opa icles
MAN 26
Maghemi e pa icles we e p epa ed by sodium bo ohyd ide (NaBH
4
) educ ion o i on chlo ide (FeCl
3
·6H
2
O).
To 1 g o FeCl
3
• 6H
2
O dissol ed in 100 mL o wa e was added wi h s i ing 0.2 g o NaBH
4
in 10 mL o 3.5 %
NH
3
. A e hyd ogen elease, he mix u e was hea ed o 2 h a 100 °C and le on s i e o e nigh . The p oduc
was collec ed by magne and washed 5 imes wi h wa e . P oduc was dispe sed in wa e (150 mL), 100 mg
o MWCNT was added and agi a ed on Biosan OS-10 o 2 h ollowed by addi ion o 1mL o hyalu onic acid
(1 g HA/50 mL), s i ed 2 hou s and sepa a ed wi h magne . Finally, p oduc was washed wi h wa e and d ied
a 40 °C. P oduc weigh was 0.3 g.
3. RESULTS AND DISCUSSION
Figu e 1
Cha ac e iza ion o pa amagne ic pa icles called MAN 26. (A) X- ay luo escence elemen al
analysis, showing elemen al composi ion o pa amagne ic mic opa icles. (B) SEM scan o pa amagne ic
mic opa icle in esolu ion o 1 µm, showing he pa icle size. (C) SEM scan o pa amagne ic
mic opa icle mic opa icle su ace in esolu ion o 200 nm. (D) IELC esul s showing abili y
o pa amagne ic mic opa icles bound equi ed subs ances speci ically. (E) SECM scan showing ela i e
cu en esponse o pa amagne ic mic opa icle wi hou aline bound. (F) SECM scan o pa amagne ic
mic opa icle wi h aline bound showing inc eased ela i e cu en esponse.
Aim o ou s udy was p ima ily isola ion and 2D sepa a ion o he b anched chain amino acids (BCAAs), based
on he adso p ion on he pa amagne ic mic opa icles and subsequen de e mina ion using ion-exchange liquid
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ch oma og aphy (IEC). We modi ied maghemi e wi h MWCNT and hyalu onic acid, and hese PMPs showed
excellen p ope ies o binding o aline ( eco e y 31.82 %) (Figu e 1D). We used B i on-Robinson bu e
wi h pH 2 which causes (BCAAs) p o ona ion ha leads in o a posi i e cha ging o molecules due o i s pI =
5.97 o aline, pI = 5.98 o leucine and pI = 10.76 o a ginine. In e ac ion be ween su ace o magne ic
mic opa icles and posi i ely cha ged molecules p o ides he binding be ween hem. These in e ac ions
depend on isoelec ic poin s o amino acids, which a e in his mode beha ing as he ion-exchange s. O he
amino acids and ipep ides such as GSH and GSSG, showed also ela i e good eco e ies (a ginine eco e y
15.1 %, leucine 22 %, sa cosine 0.9 %, GSSG 0.5 %, GSH 0.45 %, se ine 0.88 %).
A e con i ma ion o capabili y o bind amino acids aline, leucine and a ginine, we ca ied ou a di e en
cha ac e iza ion o PMPs. P ima ily cha ac e iza ion was XRF analysis p o iding in o ma ion abou elemen
composi ion o mic opa icles. In he Figu e 1A can be seen ha i on was de e mined o be he mos
abundan elemen (Fe ep esen ed 86.24 %), cobal (Co ep esen ed 0.2153 %) and magnesium
(Mg ep esen ed 0.0115 %). In Figu e 1B can be seen a mic og aph exp essing mic opa icles su ace and
mo phology in esolu ion o 1 µm o MAN 26 and in Figu e 1C is SEM mic og aph o PMP su ace in esolu ion
o 200 nm. On hese mic og aphs, MWCNTs a e well seen oge he wi h some smalle pa icles (in nm
dimensions) o maghemi e on hei su ace, bu mos o maghemi e o ms agg ega es wi h MWCNTs on i s
su ace. Al hough, he e a e some nanopa icles o maghemi e, i is be e o speak abou mic opa icles ha
we e o med a e d ying p ocess and used o expe imen s. In Figu es 1E, F can be seen 3D images,
exp essing a ela i e cu en esponse o PMP su ace wi hou aline and wi h aline bound.
4. CONCLUSION
In ou s udy, we syn hesized new pa amagne ic mic opa icles able o bind b anched chain amino acids as
possible ma ke s o obesi y. Leucine and aline a e impo an ma ke s o diagnosis o diabe es melli us ype
2 and a ginine is possible ma ke o me as a ic umo g ow h. The pa amagne ic mic opa icles (MAN 26) ha e
po en ial o be e isola ion om he samples o plasma, cells, bac e ia’s cells o issue and in u u e can se e
o applica ion as a pla o m o deli e y sys em and/o applica ion as a biosenso .
ACKNOWLEDGEMENTS
The inancial suppo om NAZV QK1720349 is highly acknowledged. QK1720349 "Nanopa icles
zinc as an al e na i e o an ibio ics in pigs"
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