CLASSIFICATION OF ALPHA(2)-MACROGLOBULIN-CYTOKINE INTERACTIONS BASED ON AFFINITY OF NONCOVALENT ASSOCIATION IN SOLUTION UNDER APPARENT EQUILIBRIUM CONDITIONS

Alpha2-macroglobulin (alpha2M) binds numerous cytokines; however, sinc e binding affinities have not been determined, it is difficult to comp are various alpha2M-cytokine interaetions or predict whether alpha2M-c ytokine complexes will form in the presence of other cytokine-binding macromolecules. In this investigation, we used a novel method to demon strate that transforming growth factor-beta1 (TGF-beta1), TGF-beta2, n erve growth factor-beta (NGF-beta), platelet derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and basic fibrobla st growth factor (bFGF) reversibly associate with alpha2M-methylamine to form noncovalent complexes. Apparent equilibrium was achieved in le ss than 15 min. Noncovalent alpha2M-cytokine complexes were converted into covalent complexes; however, this occurred slowly. Therefore, a r apid equilibrium assumption was applied and equilibrium dissociation c onstants were determined using a single binding site model. K(D) value s for the binding of cytokines to alpha2M-methylamine varied by 2 orde rs of magnitude. The rank order of affinity was TGF-beta2 (13 +/- 2 nm ) > TGF-beta1, NGF-beta > PDGF-BB greater-than-or-equal-to bFGF > TNF- alpha. Native alpha2M bound TGF-beta1, TGF-beta2, NGF-beta, PDGF-BB, a nd TNF-alpha. Interferon-gamma did not bind to native alpha2M or alpha 2M-methylamine. Each cytokine bound native alpha2M with lower affinity than alpha2M-methylamine except for TGF-beta2 which bound both forms with equal affinity. In non-equilibrium systems, alpha2M-methylamine a ppeared to bind more TGF-beta2 due to the more rapid dissociation of T GF-beta2-native alpha2M complex. The classification of alpha2M-cytokin e complexes according to binding affinity should predict which complex es are most likely to form in cell culture and under various condition s in vivo.