Co esponding au ho : Rusudan U idia
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N-glycosyla ion o p o eins
Nino Gu am, Ka kashadze 1, Rusudan Zu ab, U idia 1, *, Nana P oko , Tse odze 1, Leila Ta iel, Ta iash ili 1,
Sophia Anzo , G idani 2, Nona Tamaz, Bolk adze 3 and Lipa i A andil, Dolidze 1
1 Depa men o Chemical Ecology, P. Melikish ili Ins i u e o Physical and O ganic Chemis y, I . Ja akhish ili Tbilisi S a e
Uni e si y, Geo gia.
2 Depa men o Clinical Ana omy and Expe imen al Modeling, Al. Na ish ili Ins i u e o Mo phology, I . Ja akhish ili Tbilisi
S a e Uni e si y, Geo gia.
3 Facul y o Chemical Technology and Me allu gy, Geo gian Technical Uni e si y, Geo gia.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(01), 3570-3575
Publica ion his o y: Recei ed on 14 Ma ch 2025; e ised on 21 Ap il 2025; accep ed on 23 Ap il 2025
A icle DOI: h ps://doi.o g/10.30574/wja .2025.26.1.1348
Abs ac
Mailla d eac ion includes in e ac ion o educing suga wi h any compound con aining ee amino g oup, which is
esul ed in o ma ion o colo ed high-molecula compounds [1, 2]. Despi e in ensi e esea ch, a comple e mechanism
o Mailla d eac ion is s ill unde ined. P ima y p oduc o eac ion be ween suga s and amino acids, N-glycoside igh
a e i s o ma ion expe iences wo ypes o ans o ma ions: 1. hyd olyza ion in o ini ial p oduc s and 2.
ea angemen acco ding o scheme o e ed by Amado i, which p oduc is 1-amino-1-deoxy-2 ke ose [3].
We ha e s udied p o eins N-glycosyla ion mechanism du ing Mailla d eac ion. P o eins mainly pa icipa e in Mailla d
eac ion in he o m o hei ee amino g oups, o which ε-amino g oups o lysin and α-amino g oups o e minal amino
acids belong.
Keywo ds: Mailla d eac ion; N-glycoside; Amino g oups; P o eins.
1. In oduc ion
Mailla d eac ion includes in e ac ion o educing suga wi h any compound con aining ee amino g oup, which is
esul ed in o ma ion o colo ed high-molecula compounds [1, 2].
The mechanism o Mailla d eac ion was s udied i s by Hodge, who no ed a huge complexi y o in e ac ion be ween
suga s and amins and di e si y o ans o ma ions pa icipa ing in his p ocess [3].
Reac ion a e and na u e o o med compounds a e mainly de e mined by eac ion condi ions including chemical
p ope ies o ca bohyd a e and amine pa icipa ing in eac ion, eac ion medium cha ac e is ics (pH and wa e con en ,
p esence o oxygen and me al ions), empe a u e and p ocess du a ion, p esence o eac ion inhibi o s (e.g. educe s)
e c. [4].
Despi e in ensi e esea ch, a comple e mechanism o Mailla d eac ion is s ill unde ined. P ima y p oduc o eac ion
be ween suga s and amino acids, N-glycoside igh a e i s o ma ion expe iences wo ypes o ans o ma ions: 1.
hyd olyza ion in o ini ial p oduc s and 2. ea angemen acco ding o scheme o e ed by Amado i, which p oduc is 1-
amino-1-deoxy-2 ke ose [3].
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We ha e s udied p o eins N-glycosyla ion mechanism du ing Mailla d eac ion. P o eins mainly pa icipa e in Mailla d
eac ion in he o m o hei ee amino g oups, o which ε-amino g oups o lysin and α-amino g oups o e minal amino
acids belong. In he p ocess o ood p oduc ion indus ial p ocessing and s o age Mailla d eac ion is a main eason o
s uc u al and chemical change o p o eins [5]. Nu ien and unc ional p ope ies o die a y p o eins depend hea ily
on his eac ion. As a esul o Mailla d eac ion, in e molecula co alen bonds may o m be ween p o ein molecules,
i.e. c oss-linking o p o ein molecules may occu which leads o loss o nu i ional alue o he la e , in addi ion, is
causes ood quali y deg ada ion and sa e y le el dec ease [6].
Unde Mailla d eac ion condi ions, use o labeled compounds du ing in es iga ion o in e ac ions be ween p o eins
and glucose p o ides signi ican ad an age, since he p ocess can be con olled no only acco ding o ee numbe o
eac ing componen s, bu by means o labeled componen s en e ing in o eac ion.
2. Expe imen al pa
Some pa ame e s o Mailla d eac ion be ween p o ein and aldoses ha e been s udied by us on he example o bo ine
se um albumen glycosyla ion, when a glucose (1-6 14C -D-glucose wi h a speci ic adioac i i y 909,1 Becque el/ml.
mole) labeled wi h adioac i e ca bon has been aken as a glyca ing agen . Bo ine se um albumen is accessible in he
o m o pu e homogenic p o ein and is widely used in biochemical expe imen s. Ou in e es o he men ioned p o ein
has been s ipula ed by he ac ha i con ains ee amino g oup in especially la ge amoun s. In pa icula , an albumen
molecule (M = 67000) con ains 57 lysine- and 1 e minal ee amino g oup.
Wi h he pu pose o N-glycosyla ion, 1-6 14C -D-glucose has been added o albumen. We ha e aken 9,36 mg o 46,8 mg
o glucose pe 60 mg o albumen, eac ion has been ca ied ou in 30 ml o 0,06M phospha e bu e (pH = 5,0; 7,0; 8,0),
in da kness, a T = 25°C; 45°C; 65°C. In speci ied in e als samples in he amoun o 5 ml ha e been aken om eac ion
mix u e, which ha e been dialyzed in special dialysis bags SERVAPOR® (which de ains p o eins wi h ≥12000 molecula
mass). Op imum du a ion o dialysis has been es ablished ia e e yday measu emen o dialysable samples
adioac i i y. Fo ma ion o N-glycosyla ed p oduc s has been con olled ia de e mina ion o adioac i i y o 1 ml o
dialyzed solu ion. In pa allel, such es s ha e been conduc ed based on albumen and unlabeled D-glucose in o de o
de e mine colo o ma ion in ensi y and amoun o ee amino acids in glycosyla ed albumen. Amoun o 1-6 14C -D-
glucose in N-glycosyla ed albumen hyd olysa e has been de e mined by au o adiog aphy [7].
We ha e de e mined amino ace yla ed albumen necessa y o ou expe imen s acco ding o he known me hod [8]. 1
mole o ob ained amino ace yla ed albumen con ained 55,6 moles o ace yl g oup. We ha e de e mined p ima y amino
g oups [9] and ace yl g oups in albumen and amino ace yla ed albumens [10].
E ec o empe a u e and albumen-glucose mola a io on N-glycosyla ion eac ion unning a pH = 7,0 is gi en in Figu e
1.
1,3,5 – equi alen p opo ion o albumen-glucose = 1:1; 2,4 - equi alen p opo ion o albumen-glucose = 1:5
Figu e 1 E ec o empe a u e and eac ing componen s’ equi alen p opo ion on i e e sible inclusion o 1-6 14C
glucose ca bon in o albumen (0,06 M phospha e bu e , pH = 7,00)
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Unde ela i ely mild eac ion condi ions (T ≥ 50°C and mode a ely abundan quan i y o glucose), albumen may
pa icipa e in Mailla d eac ion only a he expense o lysine ε-amino g oups and e minal amino g oups a ailable in i .
Molecula mass o albumen used by us consis s 67000, while a numbe o ee amino g oups equals o 58 acco ding o
ou analyses (amine ni ogen 1.2%).
Based on his ac , 1 albumen equi alen comp ises 1155 in his eac ion. Fig. 1 shows ha unde condi ions o T = 25°C
and equi alen p opo ion o albumen-glucose 1:1, du ing i s 500 hou s o incuba ion he e is i ually no inclusion o
glucose labeled ca bon in o albumen composi ion (Fig. 1, cu e 1); in case o glucose concen a ion inc ease (equi alen
p opo ion o albumen-glucose 1:5), a deg ee o i e e sible glucose inclusion subs an ially ises (Fig. 1, cu e 2),
hough inclusion deg ee in bo h cases is e y low.
Reac ion mix u e empe a u e ise up o 45°C subs an ially boos s he i e e sible inclusion p ocess (Fig. 1, cu es 3,
4), while a 65°C empe a u e he p ocess in ensi y sha ply inc eases e en in case o eagen s p opo ion 1:1 (Fig. 1,
cu e 5). Thus, empe a u e ise has much s onge e ec on Mailla d eac ion a e hen mola a io o eac ing
componen s.
The in ensi y o Mailla d eac ion be ween albumen and glucose inc eases wi h ise o eac ion medium pH, as is seen
in Fig. 2. The deg ee o labeled ca bon inclusion in albumen inc eases wi h gain in glucose concen a ion (Fig. 2, cu e
4). In ou expe imen we ha e used low glucose concen a ion, ha is why ela i ely in ense o ma ion o melanoidin
colo ing has been egis e ed a 65° empe a u e only, in neu al and basic medium. In e es ing egula i y has been
eco ded in hese expe imen s: adioac i i y measu emen s ha e showed ha a e 192-hou exposi ion wi hin 192-
240-hou in e al, deg ee o 14C inclusion in o albumen has been ne e inc eased, bu despi e his ac , he in ensi y o
melanoidin colo ing o eac ion mix u e has subs an ially inc eased.
1 – equi alen p opo ion o albumen-glucose = 1:1, pH 5,0; 2 – equi alen p opo ion o albumen-glucose = 1:1, pH 7,0; 3 – equi alen p opo ion o
albumen-glucose = 1:1, pH 8,0; 4 – equi alen p opo ion o albumen-glucose = 1:5, pH 8,0.
Figu e 2 E ec o pH and eac ing componen s’ equi alen p opo ion on i e e sible inclusion o 1-6 14C glucose
ca bon in o albumen (0,06 M phospha e bu e , 65°C)
Mailla d eac ion be ween glucose and albumen is cha ac e ized by an ini ial induc ion pe iod, which is ge ing smalle
wi h ise o eac ion empe a u e (Fig. 1 and 2). Induc ion pe iod is ollowed by a leng hy pe iod, du ing which he
deg ee o 14C inclusion in o albumen almos linea ly inc eases wi h ime (Fig. 1). P e iously conduc ed
spec opho ome ic measu emen s also ha e poin ed a he exis ence o such induc ion pe iod du ing glucose-albumen
in e ac ion. This induc ion pe iod co esponds o N-glycoside o ma ion phase. [11] N-glycosides easily hyd olyze e en
in he neu al medium, so he ini ial phase o glucose-albumen in e ac ion is e e sible: N-glycosidic bond is hyd olyzed
du ing dialysis and a glucose molecule is de ached om albumen molecule. In pa allel wi h hyd olysis p ocess, N-
glycoside expe iences Amado i ea angemen , as a esul o which amino ke ose is o med i s , and a e wa ds – many
o he compounds “ inal p oduc s o glyca ion o else glycosyla ion”, and his eac ion pe iod co esponds o i e e sible
inse ion o glucose molecule o i s agmen s in o albumen. [12]
pulses pe minu e / mg. albumen
Time, hou
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As is seen om he Fig. 2, unde gi en condi ions o N-glycosyla ion eac ion, he momen o sa u a ion is eached a e
de ini e pe iod o ime and, albumen doesn’ link up any mo e glucose. Table 1 da a show ha his sa u a ion momen
comes despi e he ac ha he e is s ill a su icien amoun o ee amino g oups le in albumen necessa y o glucose
binding.
3. Discussion o he ob ained esul s
In ou expe imen s, a minimal deg ee o N-glycosyla ion (0,6% o glycosyla ed p ima y amino g oups in albumen) has
been egis e ed unde condi ions o equi alen p opo ion o albumen-glucose = 1:1, a 25°C empe a u e and pH – 7,0,
when eac ion pe iod was 672 hou s. A maximal deg ee o N-glycosyla ion has been egis e ed a equi alen p opo ion
o albumen-glucose = 1:5, 65°C empe a u e, pH – 8,0 in case o 240-hou du a ion (Table 1). I should be no ed ha
esul ing om 40-day incuba ion (37°C, phospha e bu e , pH 7.4) o bo ine se um albumen and glucose mix u e a
p o ein was ob ained, in which he glycosyla ion deg ee o lysine ε-amino g oups comp ised 75,7% o o al amoun o
ee amino g oups [13].
Table 1 Amoun o ie 1-6 14C-D glucose and N-glycosyla ed p ima y amino g oups in albumen
Glycosyla ed albumen p epa a ion condi ions
Albumen
Albumen-glucose, equi .
p opo ion
pH
T,
0C
ime,
hou s
A ached glucose,
mg/g
Glycosyla ed p ima y amino g oups %
(acco ding o ie 1-6 14C)
1:1
7.0
25
672
1.0
0.6
1:5
7.0
25
672
1.3
0.8
1:1
7.0
45
480
7.3
4.7
1:5
7.0
45
480
11.3
7.2
1:1
7.0
65
240
94.0
60.3
1:1
7.0
65
240
103.6
66.4
1:1
8.0
65
240
106.6
68.3
1:5
8.0
65
240
113.3
72.6
Table 2 Glucose o ma ion esul ing om hyd olysis N-glycosyla ed albumen (N-glycosyla ed albumen is ecei ed
unde ollowing condi ions: equi alen p opo ion albumen-glucose = 1:5, 65°C, pH - 8.0, 240 hou s)
Hyd olyzing agen
Tempe a u e, °C
Time, hou s
Glucose, %
0.1 N HCI
25
24
0
0.1 N HCI
25
48
ace
0.1 N HCI
100
9
0.5
1 N HCI
100
12
2.8
1N HCI
100
24
4.3
6 N HCI
100
24
0.7
As a esul o ee amino g oups’ blocking a p o ein loses he glucose binding abili y. We ha e implemen ed ace yla ion
o ee amino g oups a ailable in albumen and ha e s udied he abili y o ace yla ed p o ein compound wi h ace yla ed
p o ein. Analysis has showed ha 1 mole o ace yla ed albumen con ained 55.6 moles o ace yl g oups. Thus, almos
95% o ee amino acids ha e been blocked. In o de o elimina e hyd oly ic de achmen o ace yl g oups, he
glycosyla ion p ocess o ace yla ed albumen has been s udied by us in neu al medium, a 45°C and 65°C empe a u es.
I u ned ou ha i e e sible inclusion o glucose molecules o i s agmen s in o albumen doesn’ occu unde hese
condi ions.
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S udy o p oduc s o acidic hyd olysis o albumen N-glycosyla ion p oduc s has showed ha a glycolyzed albumen
con ains a pa o glucose in hyd olyzed o m, hough as can be seen, he basic pa o labeled glucose is i e e sibly
included in o albumen in he o m o di e en p oduc s o included glucose’s ans o ma ion (Table 2).
Rega ding he da a ep esen ed in Table 2, one mus ake in o accoun ha a glucose in ensely ans o ms when hea ed
in s ongly acid medium. P obably, his ac explains i s small amoun in hyd olysa e ob ained wi h he use o 6 N HCl.
4. Conclusions
I is es ablished, ha in he p o ein glycosyla ion p ocess (bo ine se um albumen ([1-6 14C] – D-glucose)) he eac ion
in ensi y inc eased wi h g ow h o eac ion medium pH. Deg ee o labeled ca bon inclusion in o albumen inc eases wi h
g ow h o glucose concen a ion. Reac ion is cha ac e ized by ini ial induc ion pe iod, which educes wi h eac ion
empe a u e ise. Induc ion pe iod is ollowed by a leng hy pe iod, du ing which a deg ee o 14C inclusion in o albumen
almos linea ly inc eases wi h ime. A e de ini e pe iod o ime he momen o sa u a ion is eached and despi e
a ailabili y in albumen o su icien amoun o ee amino g oups necessa y o glucose binding, albumen no mo e links
up any mo e glucose.
Resul ing om ee amino g oups blocking (ace yla ed albumen) p o ein loses he glucose a achmen abili y. N-
glycosyla ed albumen con ains pa o glucose in hyd olyzed o m, bu he main pa o labeled glucose is i e e sibly
included in o albumen, in he o m o di e en p oduc s o glucose ans o ma ion.
Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
No con lic o in e es o be disclosed.
Re e ences
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