Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography

Abstract

A simple and reliable method for the determination of the concentration and function of alpha-2-macroglobulin (α 2 M) by zymography was developed. The method is based on the covalent binding of α 2 M and trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the electrophoretic gel. The results showed that α 2 M binds trypsin in a concentration-dependent manner exhibiting a linear relation. The sensitivity of the method is 125 nM and the intra-assay coefficient of variation 4.2 %. Freezing of α 2 M induces its partial denaturation, which could be seen as the reduction in the amount of functional molecule and its reactivity with trypsin. The reported method enables measurement of α 2 M taking into consideration both its quantity and function, stressing the importance of the determination of the amount of physiologically active molecules and not just their presence in the sample. The method was further confirmed using α 2 M from patients with end-stage renal disease who are known to be under increased oxidative stress and inflammation, which are expected to modify the structure of proteins.