Fragmentation of a mollusc haemocyanin with plasmin and immunological identification of the fragments

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Abstract
Haemocyanin from the gastropod mollusc Lymnaea stagnalis (pond snail) was partially digested with plasmin under a variety of experimental conditions and the products of digestion analysed by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Fragments were obtained corresponding to one, two, three, four and five oxygen-binding domains, one domain having a mol.wt. of approx. 50000 and containing 2 ions of Cu. The fragments obtained after extensive digestion were non-identical immunologically, and summation of their molecular weights allowed a minimal mol.wt. of 413000 to be calculated for the original, undigested, eight-domain polypeptide chain. The use of mild-digestion conditions allowed the time course and sequence of the digestion to be monitored. An initial cleavage gave a three-domain and a five-domain fragment. The three-domain fragment was resistant to further digestion. The five-domain fragment could be digested further to give, successively a four-domain, a three-domain, and finally a two-domain fragment, single-domain units being cleaved. These data form the basis for a proposed sequence for the different domains in the original chain.