PROTEASE ACTIVITY IN COCKROACH AND BASIDIOMYCETE ALLERGEN EXTRACTS

Inherent proteolytic activity was estimated in cockroach and basidiomy cete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Low ry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 24 8 (Trypsin, 1.0 U), and 533 mug/ml (Pronase, 0.5 U) of soluble peptide s. American whole body cockroach extract (0.1 mg dry weight) released 330 mug/ml of soluble peptides, representing 13 trypsin equivalent uni ts (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 mug/ml (0.9 TEU/mg) and Pleurotus cap extract released 11 2 mug/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocyb e cubensis and spores of Psilocybe and the puffball Calvatia cyathifor mis showed negligible amounts of proteolytic activity. The protease in hibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhib itor ethylene diaminetetraacetic acid inhibited the proteolytic activi ty of Pleurotus spores by 95% (@1 mM). Loss of allergen activity as de termined by RAST inhibition and immunoprinting correlated with proteas e activity. Thus, in the preparation and handling of allergen extracts , one should employ conditions that minimize proteolysis.