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 (α2M) by zymography was developed. The 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 showed that α2M 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 α2M 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 α2M 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 α2M 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. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173042]

Keywords

• alpha-2-macroglobulin
• functional assay
• electrophoresis