Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with I % bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K. 0.1 U), 248 (Trypsin, 1.0 U), and 533 /lg!ml (Pronase, 0.5 V) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 /lg!ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extractsfrom spores of the mushroom Pleurotus ostreatus released 230 /lg!ml (0.9 TEV!mg) and Pleurotus cap extract released 112 /lg!ml (0.5 TEU!mg). Mycelium ofPleurotus and the mushroom Psilocybecubensis and spores ofPsilocybe and the pujJball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonylflouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@/ mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@I mM). Loss of allergen activity as determined by RAST inhibition and immunoprinting correlated with protease activity. Thus, in the preparation and handling of allergen extracts, one should employ conditions that minimize proteolysis. (Allergy Allergy Proc.M ostallergens that have been characterized to date, are globular proteins susceptible to degradation by proteases. 1.2 Allergenic extracts usually contain numerous proteins and include several allergens. Some proteins contained in allergen extracts may be proteases that degrade allergens. 3 -7 Indeed, the major dust mite allergen is, itself a protease. 8 The presence of protease susceptible allergens in a mixture (allergen extract) with proteases implies an inherently unstable preparation.Allergen degradation by proteases reduces the quality of an extract, either for the clinic or laboratory. Inherent proteases that degrade extracts impede research efforts to characterize/standardize extracts, and present potential quality control problems to extract manufacturers. Degraded or poor quality extracts also compromise clinical practice because extracts must maintain potency for extended times. Extracts are also commonly mixed in clinical settings, and proteases in one extract can destroy activity in other extracts.The purpose of this study is to assess inherent protease activity in cockroach and basidiomycete extracts, examine the effect of these proteases on allergens, and evaluate inhibitors that arrest proteolytic allergen degradation. MATERIALS AND METHODS Patient SeraS era were obtained from atopic subjects (positive skin tests to common inhalants and a personal and/or family history of allergy) with symptoms of rhinitis and/or asthma. Equal volumes of individual sera were pooled for use in RAST inhibition and immunoprinting. A control pool was prepared from sera of atopic patients that were skin test and RAST negative to American Whole Body Cockroach Extract (AWBE) or basidiomycete extracts. 263
Calvatia cyathiformis allergens in unfractionated extract (crude), and in extract sequentially fractionated by gel filtration (GF) and hydrophobic interaction chromatography (HIC) were tested for stability. C. cyathiformis allergen sources (crude, GF, HIC) were sampled immediately (0 h), or incubated at 4, 24 or 37 °C and then sampled at 8, 24 or 96 h. Polyacrylamide gel-isoelectric focusing immunoprints revealed 3 allergen(s) groups, or bands (Bds) with respective pi of 3.6–4.6, 6.6 and 9.3. Only Bds 3.6–4.6 were stable at 37 °C. At 24°C, Bds3.6–4.6 persisted to 96 h, Bd6.6 persisted 24 h, and Bd9.3 waned in 8 h. At 4°C all 3 allergens in HIC were stable for 8 and 24 h; Bd9.3 was reduced at 96 h. All allergen activity was labile to low pH conditions except for Bds3.6–4.6. Proteinase K degraded Bd9.3 more rapidly than Bd6.6. Immunoprint patterns corresponded to the stained gels and were consistent among different sources. Bd9.3 is very labile, but reactive with 63% of sera tested. Since 10–15% of C. cyathiformis reactors bind IgE only to Bd9.3, this is notable variability, and significant for diagnosis and treatment.
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