Relaxometric determination of binding between Mn(II)-UDP and Mn(II)-UDP-glucose in aqueous solution
Full text
Ou e e ence: CAR 6348 P-au ho que y- 11
AUTHOR QUERY FORM
Jou nal: CAR
A icle Numbe : 6348
Please e-mail o ax you esponses and any co ec ions o:
E-mail: co ec ions.essd@else ie .sps.co.in
Fax: +31 2048 52799
Dea Au ho ,
Please check you p oo ca e ully and ma k all co ec ions a he app op ia e place in he p oo (e.g., by using on-sc een anno a ion in he PDF
file) o compile hem in a sepa a e lis . No e: i you op o anno a e he file wi h so wa e o he han Adobe Reade hen please also highligh
he app op ia e place in he PDF file. To ensu e as publica ion o you pape please e u n you co ec ions wi hin 48 hou s.
Fo co ec ion o e ision o any a wo k, please consul h p://www.else ie .com/a wo kins uc ions.
Any que ies o ema ks ha ha e a isen du ing he p ocessing o you manusc ip a e lis ed below and highligh ed by flags in he p oo . Click
on he ‘Q’ link o go o he loca ion in he p oo .
Loca ion in
a icle
Que y / Rema k: click on he Q link o go
Please inse you eply o co ec ion a he co esponding line in he p oo
Q1 Please con i m ha gi en names and su names ha e been iden i ied co ec ly.
Thank you o you assis ance.
Please check his box i you ha e no
co ec ions o make o he PDF ile
G aphical abs ac
pp xxx–xxxRelaxome ic de e mina ion o binding be ween Mn(II)–UDP and Mn(II)–UDP-glucose in aqueous solu ion
E elka Fa kas
*
, O solya Szabó, Gyula Ti csó, László Somsák
*
O
HO
HO
HO
HO
OPOPO
O
OHHO
O O
O O N
NH
O
O
Mn2+
O
HO
HO
HO
HO
OPOPO
O
OHHO
O O
O O N
NH
O
O
Mn
+
log K=2.98
Δ
G = -4.07 kcal/mol
CAR 6348 No. o Pages 1, Model 5G
28 Decembe 2012
1
Relaxome ic de e mina ion o binding be ween Mn(II)–UDP and
Mn(II)–UDP-glucose in aqueous solu ion
E elka Fa kas
a,
⇑
,O solya Szabó
a
,Gyula Ti csó
a
,László Somsák
b,
⇑
a
Depa men o Ino ganic and Analy ical Chemis y, Uni e si y o Deb ecen, H-4010 Deb ecen, PO Box 21, Hunga y
b
Depa men o O ganic Chemis y, Uni e si y o Deb ecen, H-4010 Deb ecen, PO Box 20, Hunga y
a icle in o
A icle his o y:
Recei ed 29 Sep embe 2012
Recei ed in e ised o m 28 No embe 2012
Accep ed 30 No embe 2012
A ailable online xxxx
Keywo ds:
20 Relaxome y
S abili y cons an
Manganese(II)
UDP-glucose
Complex
abs ac
The applicabili y o elaxome y o he de e mina ion o o ma ion cons an s o Mn(II)–UDP
(logK= 3.78) and Mn(II)–UDP-glucose (logK= 2.98) complexes is demons a ed. The ob ained alue
indica es a well-defined in e ac ion be ween Mn(II) and UDP-glucose in aqueous solu ion (pH = 5.50)
wi h
D
G= –4.07 kcal/mol.
Ó2012 Else ie L d. All igh s ese ed.
1. In oduc ion
Glycosyl ans e ases ca alyze he biosyn hesis o glycosidic
linkages o p oduce oligo- and polysaccaha ides as well as a wide
a ie y o o he na u al p oduc s by conjuga ing suga s o lipids,
p o eins, nucleic acids, an ibio ics, o se e al ypes o o he small
molecules.
1
The so-called Leloi - ype enzymes use suga diphos-
40
phonucleo ides (NDP-suga s, e.g., UDP-glucose 1) as hei glycosyl
dono subs a es. In hese de i a i es he suga moie y, oge he
wi h he accep o , is esponsible o he specifici y o he eac ion
while he py ophospha e ac s as a lea ing g oup and also as a che-
la o o he co ac o me al ion (usually Mg(II) o Mn(II)) in mos o
he GT-A old s uc u es.
2
The me al ion acili a es depa u e o he
nucleoside diphospha e by s abilizing he de eloping nega i e
cha ge as isualized by a simplified ep esen a ion o a compu ed
model o he ansi ion s a e (2) o a eac ion ca alyzed by an
in e ing glycosyl ans e ase.
3
50
O
HO
HO
HO
HO
OPOPO
O
OHHO
OH OH
O O N
NH
O
O
ð1Þ
O
HO
HO
HO
HO
O
POPO
O
OHHO
O O
OO
M2+
O-Accep o
M = Mg o Mn
OO
H
δ−
δ+
δ+
δ+
δ−N
NH
O
O
ð2Þ
Some da a on he in e ac ion o Mn(II) and UDP-suga s we e e-
po ed in he se en ies. Thus, om e alua ion o ESR i a ion o
Mn(II) wi h UDP-galac ose a K
diss
= 14.5 ± 1.1 mM alue (pH 8.0,
0.08 M N-me hylmo pholine (NMM) con aining 0.08 M KCl a
26 ± 2 °C) was ob ained.
4
In ha pape
4
aK
diss
19 mM ob ained
60
o Mn(II)–UDP-glucose om p o on elaxa ion enhancemen
expe imen s was ci ed om Re .
5
In ano he pape e e ence was
0008-6215/$ - see on ma e Ó2012 Else ie L d. All igh s ese ed.
h p://dx.doi.o g/10.1016/j.ca es.2012.11.026
⇑
Co esponding au ho s. Tel.: +36 52512900x22306; ax: +36 52512660 (E.F.);
el.: +36 52512900x22348; ax: +36 52512744 (L.S.).
E-mail add esses: [email p o ec ed] (E. Fa kas), [email p o ec ed].
hu (L. Somsák).
Q1
Ca bohyd a e Resea ch xxx (2012) xxx–xxx
Con en s lis s a ailable a SciVe se ScienceDi ec
Ca bohyd a e Resea ch
jou nal homepage: www.else ie .com/loca e/ca es
CAR 6348 No. o Pages 6, Model 5G
28 Decembe 2012
Please ci e his a icle in p ess as: Fa kas, E.; e al. Ca bohyd . Res. (2012), h p://dx.doi.o g/10.1016/j.ca es.2012.11.026
made o unpublished obse a ions s a ing ha he appa en
dissocia ion cons an o he Mn(II)–UDP-galac ose complex was
7.5 mM, howe e , de ails and ci cums ances o he de e mina ion
we e no indica ed.
6
O he ESR s udies a 18 °C a pH = 7.4 allowed
he de e mina ion o an associa ion (s abili y) cons an K= 58.3
M
1
o Mn(II)–UDP-glucose.
7
Since ha ime, o he bes o ou
knowledge, he e has been only one epo on he in e ac ion o
Mn(II) ions and diphospha e con aining molecules s udied by iso-
70
he mal i a ion calo ime y epo ing a s abili y cons an
K= 169 M
1
o Mn(II)–UDP-glucose (in 100 mM HEPES bu e ,
pH 7.5 a 37 °C, ionic s eng h unknown).
8
The da a e e ing o
he Mn(II)–UDP-glucose sys em we e con e ed in o compa able
logK(s abili y cons an ) alues which a e collec ed in Table 1
(en ies 7–9).
Gi en he a iance shown by he abo e da a we se ou o
de e mine s abili y cons an s o he complex o Mn(II) wi h
UDP-glucose by me hods o he han hose applied so a . Such da a
can be use ul in mechanis ic e alua ions o glycosyl ans e ase ca -
80
alyzed eac ions. Fo compa ison, complex o ma ion be ween
Mn(II) and UDP as a model sys em has been also in es iga ed.
2. Expe imen al
2.1. Reagen s
UDP and UDP-glucose we e pu chased om Sigma–Ald ich and
Ca bosyn h, espec i ely, and we e used wi hou u he pu ifica-
ion. The concen a ions o hei s ock solu ions we e de e mined
ia pH-po en iome y wi h he help o G an unc ions.
9
The Mn(II) s ock solu ion was p epa ed by dissol ing
MnCl
2
4H
2
O (Reanal) in i-dis illed wa e , which con ained a
90
known amoun o HCl o minimize hyd olysis and oxida ion o
he Mn(II). The Mn(II) concen a ion o he s ock solu ion was con-
fi med by g a ime ic analysis ia p ecipi a ion as MnNH
4
PO
4
H
2
O,
while pH-po en iome y was used o de e mine he acid
concen a ion.
2.2. Po en iome ic s udies
The pH-po en iome ic i a ions we e made wi h a Radiome e
pHM 93 ins umen equipped wi h a Me ohm combined elec ode
( ype 6.0234.100). The i an was added om a Me ohm 715 Dos-
ima au oma ic bu e e. The measu emen s we e ca ied ou a
100
25.0 °C and a an ionic s eng h o 0.2 M (KCl). Solu ions o HCl
and ca bona e- ee KOH (ca. 0.2 M, used as he i an ) we e p e-
pa ed om Me ck p oduc s and hei concen a ions we e de e -
mined by pH i a ions. The elec ode sys em was calib a ed
acco ding o I ing e al.
10
o con e pH eadings in o hyd ogen
ion concen a ions. The pH-po en iome ic i a ions we e pe -
o med a 2.0 6pH 611.0 (o un il p ecipi a ion occu ed). The li-
gand concen a ion was 1 10
3
M and he me al- o-ligand a io
anged om 1:1 o 1:5. The ini ial olume o he samples was
10.0 mL. The expe imen al esul s we e u ilized o es ablish he
110
s oichiome y o he species and o calcula e he s abili y con-
s an s. Species s oichiome y and s abili y cons an s we e de e -
mined wi h he compu e p og am PSEQUAD.
11
Volumes o he
i an we e fi ed and he accep ed fi ings we e always below
110
2
mL.
2.3. Relaxome y
Relaxome ic measu emen s
12
we e made on a B uke Minispec
MQ-20 ins umen ope a ing a 20 MHz. The spin-la ice elaxa ion
ime, T
1
, was measu ed wi h his echnique by he in e sion- eco -
e y me hod.
120
The elaxi i y o he Mn(II)aqua was de e mined in a sepa a e
expe imen using he published me hodology.
12
The olume o
he samples was 1 mL, and he concen a ion o he me al ion a -
ied in he ange 0.2 10
3
–20 10
3
M.
Fo he in es iga ed sys ems, he olume o he samples was
0.5 mL, and he ionic s eng h was 0.2 M KCl a 25 °C. To se he
pH NEP (N-e hyl-pipe azine wi h a logK
2
= 5.58 (0.02) a I = 1.0 M
KCl and 25 °C, pH = 5.50) and HEPES (4-(2-hyd oxye hyl)-1-pipe-
azinee hanesul onic acid, pH = 7.57) bu e solu ions we e used.
The measu emen s on Mn(II)–UDP samples we e pe o med a
130
pH = 5.50 only, while a bo h pH alues on he Mn(II)–UDP-glucose
sys em. All s udies we e ca ied ou unde an ine a mosphe e
(A ).
Fo he Mn(II)–UDP and Mn(II)–UDP-glucose sys ems he me al
ion concen a ion in he samples was se o 2 10
3
M while he
me al o ligand a io a ied in he ange o 1:(0.25–3) and
1:(0.25–6), espec i ely. Fo he Mn(II)–UDP sys em, he
pH-dependence was also s udied a 4.9 6pH 66.6 a a me al- o-li-
gand a io o 1:2. The Mn(II)–UDP-glucose sys em was also in es-
iga ed by i a ing he samples wi h Mn(II), gi ing me al- o-ligand
140
a ios a ying om he ini ial 1:5 o 5:1.
Relaxi i y alues we e calcula ed using he obse ed (1/T
1
) al-
ues and he equilib ium concen a ion o he complex calcula ed
om:
12
½MnH
x
L¼1=T
Mn
1
½Mn
1=T
0
1
1=T
Mn
1
1=T
MH
x
L
1
whe e 1/T
0
1
=1/T
1
1/T
w
and [Mn]
= [Mn] + [MnH
x
L].
1/T
w
= diamagne ic con ibu ion o he elaxa ion a e (1/T
1
in
150
he absence o Mn(II)).
Table 1
Compa ison o s abili y cons an s o Mn(II)–UDP and Mn(II)–UDP-glucose (UDP-Glc) complexes
En y Equilib ium p ocess Cons an
a
(logK) Me hod Condi ions
b
Re .
1 Mn(II) + UDP
2
= [Mn(UDP)] 4.14(5) pH-me y 25 °C, 0.2 M KCl This wo k
2 4.07 pH-me y 25 °C, 0.1 M NaNO
3
13
3 3.45 Calo ime y 37 °C, 0.1 M HEPES, pH 7.5 8
4 3.51 ESR i a ion 18 °C, pH 7.4 7
5 3.94 Unknown
c
Unknown
c
17
6 3.78 (2) Relaxome y 25 °C/0.2 M KCl, pH 5.50 This wo k
7 Mn(II) + UDP-Glc
2
= [Mn(UDP-Glc)] 2.23 Calo ime y 37 °C, 0.1 M HEPES (pH 7.5) 8
8 1.72 P o on elaxa ion enhancemen Unknown
d
5
9 1.77 ESR i a ion 18 °C, pH 7.4 7
10 2.98 (7) Relaxome y 25 °C, 0.2 M KCl, 0.05 M NEP, pH 5.50 This wo k
11 3.57 (13) Relaxome y 25 °C, 0.2 M KCl, 0.04 M HEPES, pH 7.57 This wo k
a
S anda d de ia ions in he las significan digi a e gi en in pa en heses.
b
Abb e ia ions: HEPES: 4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid, NEP: N-e hyl-pipe azine.
c
The e e ed book was una ailable o us, he e o e, he condi ions o he measu emen emained unknown. The gi en alue was ci ed in Re . 6.
d
The e e ed book was una ailable o us, he e o e, he condi ions o he measu emen emained unknown. The gi en alue was ci ed in Re . 4.
2E. Fa kas e al./ Ca bohyd a e Resea ch xxx (2012) xxx–xxx
CAR 6348 No. o Pages 6, Model 5G
28 Decembe 2012
Please ci e his a icle in p ess as: Fa kas, E.; e al. Ca bohyd . Res. (2012), h p://dx.doi.o g/10.1016/j.ca es.2012.11.026
1/T
1Mn
and 1/T
1MnHxL
a e he elaxi i ies o he Mn(II) and he
complex o med.
x= 1 Mn{H(UDP)} x= 0 Mn(UDP-glucose).
L = UDP o UDP-glucose.
The compu e p og am PSEQUAD
11
was used o ob ain he s a-
bili y cons an s om he calcula ed equilib ium concen a ions o
he complexes.
3. Resul s and discussion
160
The main goal o his wo k was o de e mine he s eng h o
in e ac ion be ween Mn(II) and UDP-glucose 1. The pH-me ic
i a ion o UDP-glucose p o ided clea e idence o dissocia ion
o a single p o on in he measu able pH- ange, which (based on
chemical e idences and li e a u e suppo
13
) belongs unambigu-
ously o he dep o ona ion o he neu al N(3)H (see Cha 1)o
he nucleobase esidue. Acco ding o his esul , he diphospha e
moie y o 1 eleases a p o on in he e y acidic egion (pH 2)
and he UDP-glucose
2
o m p edomina es om he beginning o
he measu able pH- ange. As a consequence, any pH-e ec canno
170
belong o he me al–ion complexa ion o he diphospha e esidue
and pH-po en iome y canno be applied o s udy complex o ma-
ion be ween Mn(II) and UDP-glucose 1. Owing o he e y low
in ensi y o he spin- o bidden d–d bands o he high-spin d
5
Mn(II) complexes UV– isible spec opho ome y could no be
applied o his sys em ei he .
14
Howe e , o ma ion o he
Mn(II)–UDP-glucose complexes can be ollowed by measu ing
he spin-la ice elaxa ion a e (1/T
1
) o he wa e p o ons, since
he complex o ma ion be ween he Mn(II) ion and he anionic li-
gand educes he numbe o me al-coo dina ed wa e molecules,
180
hus p o iding a use ul echnique o quan i a i e analysis o he
binding equilib ium. To check he applicabili y o his me hod o
he Mn(II)–UDP-glucose sys em, a model sys em, Mn(II)–UDP,
was s udied fi s . The dissocia ion cons an s o he species exis ing
a pH 2o UDP-glucose
2
1and UDP
2
3(Cha 1) we e de e -
mined by pH-po en iome y and he alues (which a e in e y
good ag eemen wi h he li e a u e
13
) a e shown in Cha 1.
3.1. Mn(II)–UDP model sys em
S abili y cons an s o Mn(II)–UDP ound in he li e a u e a e
lis ed in Table 1 (en ies 2–5). In his sys em he complex o ma-
190
ion is accompanied by a measu able pH-e ec , he eby, p io o
he elaxome ic measu emen s, he s abili y cons an o he
Mn(II)–UDP complex could be de e mined ia pH-po en iome y.
Rep esen a i e i a ion cu es a e shown in Figu e 1.
The calcula ed s abili y cons an o he Mn(II) + UDP
2
=
[Mn(UDP)] p ocess (Table 1,en y 1) is in good ag eemen wi h
he li e a u e alue,
13
also de e mined by pH-po en iome y (en y
2; exclusi e coo dina ion o UDP ia he diphospha e moie y was
p o en in ha pape
13
). The somewha bigge di e ence be ween
ou alue and ha de e mined by iso he mal i a ion calo ime y
8
200
(en y 3) o ESR i a ion
7
(en y 4) is possibly due o he signifi-
can ly di e en condi ions o empe a u e and ionic s eng h.
The elaxi i y o 8.14 ± 0.03 mM
1
s
1
, de e mined om an
indi idual measu emen , o Mn(II)aqua is in good ag eemen wi h
he li e a u e.
14
The elaxi i y o 9.91 ± 0.58 mM
1
s
1
ob ained o
[Mn(UDP)] was significan ly di e en , hus, he elaxi i y (wa e -
p o on elaxa ion a e) is in p inciple applicable o he de e mina-
ion o he s abili y cons an o he complex (Table 1,en y 6). A
compa ison o he s abili y cons an s ob ained by elaxome y
and by pH-po en iome y (en ies 1 and 6, espec i ely) shows
210
an accep able ag eemen , pa icula ly, i he somewha di e en
condi ions (see Sec ion 2) a e also aken in o accoun .
3.2. Mn(II)–UDP-glucose sys em
A e p o ing he applicabili y o he elaxome ic me hod o
s abili y cons an de e mina ion in he model sys em, measu e-
men s we e pe o med on he Mn(II)–UDP-glucose sys em. The
expe imen al da a could be con incingly fi ed by assuming he
exis ence o he complex [Mn(UDP-glucose)] (R
[Mn(UDP-glucose)]
=
8.82 ± 0.40 mM
1
s
1
a pH = 5.50; R
[Mn(UDP-glucose)]
= 8.91 ±
0.39 mM
1
s
1
a pH = 7.57). I is impo an o no e ha he esul
220
ob ained a pH = 7.57 ca ies a highe unce ain y as compa ed o
ha a pH = 5.50, because a highe pH dep o ona ion o N(3)H
akes place o a small ex en (ca. 2–3 %). Thus, in e molecula p o-
o opic exchange p ocesses be ween he p o ona ed and dep o o-
na ed species migh al e he elaxa ion a e o he wa e p o on
and he elaxi i y, as well. The e o e, we hink ha he esul
ob ained a pH 5.50 is mo e eliable. The loga i hmic s abili y
cons an s ob ained o he Mn(II)–UDP-glucose complex a e shown
in Table 1,en ies 10 and 11. The di e ence be ween hese alues,
appa en ly due o he change o pH, p obably eflec s he abo e
230
p ocesses.
The s abili y cons an calcula ed o he [Mn(UDP-glucose)] (en-
y 10) is lowe wi h ca. one log uni han ha o [Mn(UDP)] (en y
6). A simila di e ence be ween hese wo cons an s was also
ound by calo ime y
8
(compa e en ies 3 and 7) and by ESR i a-
ion
7
(en ies 4 and 9). This di e ence is p obably due o a dec ease
in he basici y o he diphospha e moie y upon subs i u ion o he
e minal OH-g oup by a ca bohyd a e esidue. The di ec co ela-
ion be ween he basici y o a coo dina ed diphospha e esidue
and he s abili y o he co esponding me al complex is de ailed
240
elsewhe e.
13
The s abili y o he Mn(II)–UDP-glucose complex is
mode a e, co esponding o a Gibbs ene gy change o
D
G= –4.07 kcal/mol. To demons a e his, he ex en o complex
o ma ion has been calcula ed as a unc ion o he analy ical
concen a ion o Mn(II). The solid line in Figu e 2 e e s o he
1:1 concen a ion a io o Mn(II) o UDP-glucose and shows ha
O
O
HO
HO
HO
HO
OPOPO
O
OHHO
O O
O O N
N(3)H
O
O
pK = 9.43(2)
HO POPO
O
OHHO
O O
O O N
N(3)H
O
O
pK = 6.18(7)
pK = 9.30(1)
Cha 1. UDP-glucose
2
1and UDP
2
3( he p edominan o ms o he compounds
exis ing a he beginning o he measu able pH- ange, ca. pH 2). The pKs belonging
o he neu al N(3)H esidues and he e minal OH g oup o 3a e shown wi h
s anda d de ia ions in pa en heses.
E. Fa kas e al./ Ca bohyd a e Resea ch xxx (2012) xxx–xxx 3
CAR 6348 No. o Pages 6, Model 5G
28 Decembe 2012
Please ci e his a icle in p ess as: Fa kas, E.; e al. Ca bohyd . Res. (2012), h p://dx.doi.o g/10.1016/j.ca es.2012.11.026
he ac ion o he complex ( a io o he complex o he o al con-
cen a ion o he me al ion) is significan o analy ical concen a-
ions abo e ca. 10
4
M. Fo ma ion o he complex becomes
negligible below 10
4
M (pc is abo e 4) and is p ac ically ze o a
250
and below 10
5
M. Howe e , as is clea ly shown by he dashed
line, i he Mn(II) concen a ion is dec eased only a a cons an con-
cen a ion (1.0 mM) o UDP-glucose, a well defined a io (abou
50%) o he o al Mn(II) emains complexed e en a pc
Mn(II)
=6
(1.0
l
M).
Fo physiological (in acellula ) concen a ion o Mn(II) a ious
alues can be ound in he li e a u e om 10
8
M
8
o 2–3
10
5
M,
15
while a be e conco d exis s o ha o UDP-glucose
(2–4 10
4
M).
15,16
I ollows om he abo e conside a ion ha
unde physiological condi ions o ma ion o he Mn(II)–UDP-glu-
260
cose complex is e y mino o e en negligible.
The highe s abili y o he Mn(II)–UDP complex (ac ually one o
he p oduc s o a eac ion ca alyzed by a glycosyl ans e ase)
compa ed o ha o he Mn(II)–UDP-glucose complex (one o he
subs a es o he eac ion) mus ha e implica ions o unde s and-
ing he ca aly ic mechanism. The obse ed di e ence in he s abil-
i ies ce ainly eflec s he enhanced lea ing abili y o UDP upon
Mn(II) complexa ion. Howe e , in he en i onmen o he enzyme’s
ac i e si e a mo e complica ed in e play o se e al o he ac o s
(e.g., binding o he me al ion o addi ional complexing esidues
270
like he equen DXD mo i , geome y o he complex, in e ac ions
o he ac i e si e esidues wi h o he pa s o he subs a e/p oduc ,
con o ma ional changes o he p o ein du ing he ca aly ic p ocess)
ha e o be conside ed. In his espec a e y ecen s udy sugges s
highe s abiliza ion o a glycosyl ans e ase upon simul aneous
addi ion o UDP-glucose and Mn(II) as compa ed o ha o UDP
and Mn(II).
15
Such issues need, no doub , u he expe imen al
s udies and heo e ical analyses.
In conclusion, as a esul o his wo k (i) he applicabili y o
elaxome y o he de e mina ion o he s abili y cons an o he
280
Mn(II)–UDP-glucose complex has been demons a ed; (ii) as a con-
sequence o he mode a e s abili y o ha complex, i manga-
7
9
11
pH
UDP
1:5
1:3
1:2
3
5
-1.0 0.0 1.0 2.0 3.0 4.0
Base equi alen
1:1
Figu e 1. Po en iome ic (pH) i a ion cu es o UDP () and Mn(II)–UDP a me al- o-ligand a ios o : 1:5 (j), 1:3 (N), 1:2 (x) and 1:1 (s) wi h c
ligand
=110
3
mol dm
3
.
Nega i e base equi alen s e e o acid solu ions.
0.6
0.5
0.4
]glc)]DP-g(UD
0.3
Mn-n [Mc ioF ac
0.2
F
0.1
0
3 4 4 5 5.5 635
. 4.5
pc Mn(II)
Figu e 2. F ac ion o complexed Mn(II) as a unc ion o [Mn(II)]
a 1:1 Mn(II) o UDP-glucose a io (solid line) and a cons an 1.0 mM UDP-glucose concen a ion, whe e he
a io o Mn(II) o UDP-glucose is a ied om 1:1 o 1:1000 (dashed line).
4E. Fa kas e al./ Ca bohyd a e Resea ch xxx (2012) xxx–xxx
CAR 6348 No. o Pages 6, Model 5G
28 Decembe 2012
Please ci e his a icle in p ess as: Fa kas, E.; e al. Ca bohyd . Res. (2012), h p://dx.doi.o g/10.1016/j.ca es.2012.11.026
nese(II) and UDP-glucose a e p esen a equimola concen a ion in
he sample, he complex is o med in measu able ac ion only
abo e an analy ical concen a ion o 10
5
M; (iii) a high ligand ex-
cess a la ge ac ion o Mn(II) is complexed e en a mic omola
concen a ions o he me al ion.
Acknowledgemen s
Financial suppo o his wo k was p o ided by he Hunga ian
Scien ific Resea ch Fund (OTKA CK77712) and by TÁMOP 4.2.1/B-
290
09/1/KONV-2010-0007 and TÁMOP-4.2.2./B-10/1-2010-0024 p o-
jec s co-financed by he Eu opean Union and he Eu opean Social
Fund. G.T. hanks he Hunga ian Academy o Sciences o he
awa d o a János Bolyai Resea ch Schola ship. D . Glenn He e is
hanked o linguis ic checking o he manusc ip .
Re e ences
1. Weadge, J. T.; Palcic, M. M. In Encyclopedia o Chemical Biology; Begley, P., Ed.;
Wiley, 2008; pp 198–211.
2. Lai son, L. L.; Hen issa , B.; Da ies, G. J.; Wi he s, S. G. Annu. Re . Biochem. 2008,
77, 521–555.
3003. T a oška, I. T ends Glycosci. Glyco echnol. 2005,17, 177–190.
4. Be line , L. J.; Wong, S. S. Biochemis y 1975,14, 4977–4982.
5. Dwek, R. A. Nuclea Magne ic Resonance (NMR) in Biochemis y; Cla endon P ess:
Ox o d, England, 1973. p 256.
6. Kha a, B. S.; He ies, B. G.; B ew, K. Eu . J. Biochem. 1974,44, 537–560.
7. Tsopanakis, A. D.; He ies, D. G. Eu . J. Biochem. 1978,83, 179–188.
8. Zea, C. J.; Camci-Unal, G.; Pohl, N. L. Chem. Cen . J. 2008,2, 15.
9. G an, G. Ac a Chem. Scand. 1950,4, 559–577.
10. I ing, H. M. N. H.; Miles, M. G.; Pe i , L. D. Anal. Chim. Ac a 1967,38, 475–488.
11. Zékány, L.; Nagypál, I. In Compu a ional Me hods o he De e mina ion o
310Fo ma ion Cons an s; Lege , D., Ed.; Plenum P ess: New Yo k, 1985; pp 291–
353.
12. Co es, S.; B üche , E.; Ge aldes, C. F. G. C.; She y, A. D. Ino g. Chem. 1990,29,
5–9.
13. Sajadi, S. A. A.; Song, B.; G egan, F.; Sigel, H. Ino g. Chem. 1999,38, 439–448.
14. D ahos, B.; Ko ek, J.; He mann, P.; Lukes, I.; Tó h, É. Ino g. Chem. 2010,49,
3224–3238.
15. D’U zo, N.; Mali o, E.; Biancucci, M.; Bo omley, M. J.; Maione, D.; Sca selli, M.;
Ma inelli, M. FEBS J. 2012,279, 3085–3097.
16. Huang, K. P.; Robinson, J. C. J. Biol. Chem. 1977,252, 3240–3244.
32017. Bock, R. M. In The Enzymes; Boye , P. D., La dy, H., My bäck, K., Eds.; Academic
P ess: New Yo k, 1960; pp 3–38.
E. Fa kas e al./ Ca bohyd a e Resea ch xxx (2012) xxx–xxx 5
CAR 6348 No. o Pages 6, Model 5G
28 Decembe 2012
Please ci e his a icle in p ess as: Fa kas, E.; e al. Ca bohyd . Res. (2012), h p://dx.doi.o g/10.1016/j.ca es.2012.11.026