Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study

A alpha M-2 homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep alpha M-2 was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep alpha M-2 was found to be made up of two fragments of 102 and 31 kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79 angstrom, which makes it much more compact than its human homologue. It entraps only 1 mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures - enthalpy change (Delta H), entropy change (Delta S) and Gibb's free energy change (Delta G), along with number of binding sites (N) and affinity constant (K) are explored for alpha M-2-trypsin binding for the first time for any known alpha M-2 molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin-alpha M-2 interaction is enthalpy driven event. (C) 2015 Elsevier B.V. All rights reserved.