Comparison of Limulus α-macroglobulin with human α₂-macroglobulin: Thiol ester characterization, subunit organization, and conformational change

The properties of the thiol ester-containing α-macroglobulin (αM) from the horseshoe crab (Limulus polyphemus) have been compared with those of the human analogue (α₂M). Thiol ester accessibility was more restricted in Limulus αM than in human α₂M. Fluorescent probes attached to the thiol ester cysteine indicated very similar local environments in the cleaved state of the two αMs. The separation between the two thiol ester cysteines in the cleaved state, determined by fluorescence resonance energy transfer, was also very similar for the two αMs. Differences were found in the oligomerization state and conformational changes of the two proteins. Whereas human α₂M appears to be exclusively a dimer of dimers, Limulus αM can exist in both tetrameric and dimeric forms, although with marked preference for the dimer. Conformational change within a dimeric trapping unit, monitored by 6-(p- toluidino)-2-napthalene-sulfonic acid fluorescence change, showed that each monomer of the Limulus αM dimer appears to change conformation independently, whereas human α₂M requires both thiol esters within a functional unit to be cleaved before the conformational change occurs. Taken together, these findings indicate that, whereas individual thiol esters in both types of αM are similar in properties, differences in subunit-subunit interaction result both in differences in state of oligomerization and in cooperativity of conformational change, which may reflect a fundamentally different organization of the subunits within a dimer in the two αMs.