TY  - CONF
AU  - Četić, Danilo
AU  - Miljuš, Goran
AU  - Dobrijević, Zorana
AU  - Gligorijević, Nikola
AU  - Vilotić, Aleksandra
AU  - Nedić, Olgica
AU  - Penezić, Ana
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6663
AB  - Human transferrin (Tf) is a bilobal 76 kDa iron-binding glycoprotein present in human
serum. Each lobe has the ability to bind one ferric ion (Fe3+) and a single synergistic
bicarbonate anion. The main role of Tf is to transport Fe3+ ions through the circulation to
cells, via interaction with transferrin receptor (TFR) on the cell surface. Previously
described methods for Tf isolation and purification are either very time-consuming or
provide Tf of lower final purity. Here we describe a fast and simple FPLC method for the
isolation and purification of Tf from human serum. Serum samples were prepared by
precipitation, while protein purification was performed on FPLC system, using an anionexchange
column. Several different buffers at the same pH were tested. Tf purified by this
method was analyzed by Western blot, followed by immunodetection, as well as with
silver staining after SDS PAGE. Its functionality was tested with respect to iron-binding
capacity (ferozzine method) and its ability to interact with TFR by immunofluorescent
staining. The conformation of purified Tf was analyzed by recording intrinsic fluorescent
emmision spectra originating from Trp residues. The method itself is highly reproducible
(intra- and interday), easy to perform (only two steps) and fast (under an hour), yielding
98% to 99% pure Tf with all buffers. Purified Tf was shown to have retained its ironbinding
capacity, as well as the ability to interact with TFR. Purified Tf also retained its
native three-dimensional structure. Described method for the isolation and purification of
Tf is fast, simple and highly reproducible, yielding a functional Tf of high purity in its
native state while offering the flexibility of using different buffer systems. All of these
features make this protocol a method of choice for the isolation and purification of Tf on a
semi-preparative scale.
PB  - Serbian Biochemical Society
C3  - Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia
T1  - Simple two-step semi-preparative isolation and purification of transferrin from human serum
SP  - 66
EP  - 66
UR  - https://hdl.handle.net/21.15107/rcub_cer_6663
ER  - 

@conference{
author = "Četić, Danilo and Miljuš, Goran and Dobrijević, Zorana and Gligorijević, Nikola and Vilotić, Aleksandra and Nedić, Olgica and Penezić, Ana",
year = "2023",
abstract = "Human transferrin (Tf) is a bilobal 76 kDa iron-binding glycoprotein present in human
serum. Each lobe has the ability to bind one ferric ion (Fe3+) and a single synergistic
bicarbonate anion. The main role of Tf is to transport Fe3+ ions through the circulation to
cells, via interaction with transferrin receptor (TFR) on the cell surface. Previously
described methods for Tf isolation and purification are either very time-consuming or
provide Tf of lower final purity. Here we describe a fast and simple FPLC method for the
isolation and purification of Tf from human serum. Serum samples were prepared by
precipitation, while protein purification was performed on FPLC system, using an anionexchange
column. Several different buffers at the same pH were tested. Tf purified by this
method was analyzed by Western blot, followed by immunodetection, as well as with
silver staining after SDS PAGE. Its functionality was tested with respect to iron-binding
capacity (ferozzine method) and its ability to interact with TFR by immunofluorescent
staining. The conformation of purified Tf was analyzed by recording intrinsic fluorescent
emmision spectra originating from Trp residues. The method itself is highly reproducible
(intra- and interday), easy to perform (only two steps) and fast (under an hour), yielding
98% to 99% pure Tf with all buffers. Purified Tf was shown to have retained its ironbinding
capacity, as well as the ability to interact with TFR. Purified Tf also retained its
native three-dimensional structure. Described method for the isolation and purification of
Tf is fast, simple and highly reproducible, yielding a functional Tf of high purity in its
native state while offering the flexibility of using different buffer systems. All of these
features make this protocol a method of choice for the isolation and purification of Tf on a
semi-preparative scale.",
publisher = "Serbian Biochemical Society",
journal = "Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia",
title = "Simple two-step semi-preparative isolation and purification of transferrin from human serum",
pages = "66-66",
url = "https://hdl.handle.net/21.15107/rcub_cer_6663"
}

Četić, D., Miljuš, G., Dobrijević, Z., Gligorijević, N., Vilotić, A., Nedić, O.,& Penezić, A.. (2023). Simple two-step semi-preparative isolation and purification of transferrin from human serum. in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia
Serbian Biochemical Society., 66-66.
https://hdl.handle.net/21.15107/rcub_cer_6663

Četić D, Miljuš G, Dobrijević Z, Gligorijević N, Vilotić A, Nedić O, Penezić A. Simple two-step semi-preparative isolation and purification of transferrin from human serum. in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia. 2023;:66-66.
https://hdl.handle.net/21.15107/rcub_cer_6663 .

Četić, Danilo, Miljuš, Goran, Dobrijević, Zorana, Gligorijević, Nikola, Vilotić, Aleksandra, Nedić, Olgica, Penezić, Ana, "Simple two-step semi-preparative isolation and purification of transferrin from human serum" in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia (2023):66-66,
https://hdl.handle.net/21.15107/rcub_cer_6663 .

TY  - CONF
AU  - Gligorijević, Nikola
AU  - Šunderić, Miloš
AU  - Vilotić, Aleksandra
AU  - Baralić, Marko
AU  - Nedić, Olgica
PY  - 2019
UR  - http://www.bds.org.rs/download/SBS_Conference_09_2019.pdf
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7291
AB  - Alpha-2-macroglobulin (α2M) is a homotetrameric blood glycoprotein having molecular
mass of 720 kDa which acts as a general protease inhibitor 1. So far, the methods to
estimate the quantity of α2M and its activity were separate procedures. The quantity is
usually measured by immunochemical assays and the anti-protease activity of α2M by
measuring the activity of trypsin bound to α2M using chromogenic substrate BAPNA 2. A
simple and reliable method for determination of the concentration and function of α2M by
zymography was developed. This method is based on the covalent binding of α2M and
trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the
electrophoretic gel. The results have shown that α2M binds trypsin in a linear,
concentration-dependent manner. The sensitivity of the method is 125 nM with an intraassay
coefficient of variation 4.2 %. Freezing of α2M induces its partial denaturation,
which can be seen as the reduction in the amount of functional molecule and its reactivity
with trypsin. The method was further tested using α2M from patients with an end-stage
renal disease who are known to be under an increased oxidative stress and inflammation,
which are expected to modify the structure of proteins. Using α2M from these patients,
lower affinity of α2M towards trypsin was detected when compaired to α2M isolated from
healthy persons. The reported zymographic method enables measurement of α2M taking
into consideration both its quantity and function, stressing the importance of determination
of the amount of physiologically active molecules and not just their total amount present in
the sample. Monitoring of the relation quantity/activity becomes very important when the
sample originates from an individual exposed to a stress or with a disease accompanied by
post-translational modifications of proteins such as diabetes, renal disease or cancer 3.
Presented method also enables determination of α2M in the presence of different modifying
chemical substances.
PB  - Serbian Biochemical Society
C3  - Serbian Biochemical Society Ninth Conference with international participation,  “Diversity in Biochemistry”, 14-16.11.2019, University of Belgrade – Kolarac Endowment, Belgrade, Serbia
T1  - Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography
SP  - 98
EP  - 98
UR  - https://hdl.handle.net/21.15107/rcub_cer_7291
ER  - 

@conference{
author = "Gligorijević, Nikola and Šunderić, Miloš and Vilotić, Aleksandra and Baralić, Marko and Nedić, Olgica",
year = "2019",
abstract = "Alpha-2-macroglobulin (α2M) is a homotetrameric blood glycoprotein having molecular
mass of 720 kDa which acts as a general protease inhibitor 1. So far, the methods to
estimate the quantity of α2M and its activity were separate procedures. The quantity is
usually measured by immunochemical assays and the anti-protease activity of α2M by
measuring the activity of trypsin bound to α2M using chromogenic substrate BAPNA 2. A
simple and reliable method for determination of the concentration and function of α2M by
zymography was developed. This method is based on the covalent binding of α2M and
trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the
electrophoretic gel. The results have shown that α2M binds trypsin in a linear,
concentration-dependent manner. The sensitivity of the method is 125 nM with an intraassay
coefficient of variation 4.2 %. Freezing of α2M induces its partial denaturation,
which can be seen as the reduction in the amount of functional molecule and its reactivity
with trypsin. The method was further tested using α2M from patients with an end-stage
renal disease who are known to be under an increased oxidative stress and inflammation,
which are expected to modify the structure of proteins. Using α2M from these patients,
lower affinity of α2M towards trypsin was detected when compaired to α2M isolated from
healthy persons. The reported zymographic method enables measurement of α2M taking
into consideration both its quantity and function, stressing the importance of determination
of the amount of physiologically active molecules and not just their total amount present in
the sample. Monitoring of the relation quantity/activity becomes very important when the
sample originates from an individual exposed to a stress or with a disease accompanied by
post-translational modifications of proteins such as diabetes, renal disease or cancer 3.
Presented method also enables determination of α2M in the presence of different modifying
chemical substances.",
publisher = "Serbian Biochemical Society",
journal = "Serbian Biochemical Society Ninth Conference with international participation,  “Diversity in Biochemistry”, 14-16.11.2019, University of Belgrade – Kolarac Endowment, Belgrade, Serbia",
title = "Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography",
pages = "98-98",
url = "https://hdl.handle.net/21.15107/rcub_cer_7291"
}

Gligorijević, N., Šunderić, M., Vilotić, A., Baralić, M.,& Nedić, O.. (2019). Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography. in Serbian Biochemical Society Ninth Conference with international participation,  “Diversity in Biochemistry”, 14-16.11.2019, University of Belgrade – Kolarac Endowment, Belgrade, Serbia
Serbian Biochemical Society., 98-98.
https://hdl.handle.net/21.15107/rcub_cer_7291

Gligorijević N, Šunderić M, Vilotić A, Baralić M, Nedić O. Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography. in Serbian Biochemical Society Ninth Conference with international participation,  “Diversity in Biochemistry”, 14-16.11.2019, University of Belgrade – Kolarac Endowment, Belgrade, Serbia. 2019;:98-98.
https://hdl.handle.net/21.15107/rcub_cer_7291 .

Gligorijević, Nikola, Šunderić, Miloš, Vilotić, Aleksandra, Baralić, Marko, Nedić, Olgica, "Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography" in Serbian Biochemical Society Ninth Conference with international participation,  “Diversity in Biochemistry”, 14-16.11.2019, University of Belgrade – Kolarac Endowment, Belgrade, Serbia (2019):98-98,
https://hdl.handle.net/21.15107/rcub_cer_7291 .