Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma.

Crayfish plasma was found to contain a proteinase inhibitor, which was purified to apparent homogeneity by acid precipitation, affinity chromatography on concanavalin A-Sepharose and hydrophobic-interaction chromatography. The inhibitor is a monomeric protein with an Mr of about 155,000 and a pI in the range 4.6-4.8. It is heat-stable and tolerant to low pH. It inhibits the serine proteinases trypsin and chymotrypsin, but not thrombin or subtilisin. Furthermore, it is efficient in decreasing the activity of a proteinase from crayfish haemolymph that is involved in the activation cascade of pro-phenol oxidase and can also block pro-phenol oxidase activation by this serine proteinase. This cascade is believed to play a central role in the recognition mechanism of non-self material in crustaceans and insects. The data presented give some evidence that the new proteinase inhibitor is involved in the regulation of this system.