Localisation of the major reactive lysine residue involved in the selfcrosslinking of proteinase‐activated Limulusα2‐macroglobulin

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Abstract
When α 2-macroglobulin (α 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other α-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other α-macroglobulins extensively form crosslinks to the reacting proteinase, Limulus α 2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus α 2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.