@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"
}
