Wongtim S, Lehrer S B, Salvaggio J E, Horner W E
Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana.
Allergy Proc. 1993 Jul-Aug;14(4):263-8. doi: 10.2500/108854193778811946.
Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 248 (Trypsin, 1.0 U), and 533 micrograms/ml (Pronase, 0.5 U) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 micrograms/ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 micrograms/ml (0.9 TEU/mg) and Pleurotus cap extract released 112 micrograms/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocybe cubensis and spores of Psilocybe and the puffball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@1 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.
通过定量测定与1%牛血清白蛋白孵育提取物后释放的酸溶性肽来评估蟑螂和担子菌提取物中的固有蛋白水解活性，采用Lowry法（西格玛公司）测定。参考蛋白酶释放出740（蛋白酶K，0.1 U）、248（胰蛋白酶，1.0 U）和533微克/毫升（链霉蛋白酶，0.5 U）的可溶性肽。美洲蟑螂全身提取物（0.1毫克干重）释放出330微克/毫升的可溶性肽，相当于13个胰蛋白酶当量单位（TEU）/毫克。平菇孢子提取物释放出230微克/毫升（0.9 TEU/毫克），平菇菌盖提取物释放出112微克/毫升（0.5 TEU/毫克）。平菇菌丝体、裸盖菇以及裸盖菇孢子和马勃的蛋白酶活性可忽略不计。蛋白酶抑制剂苯甲基磺酰氟使美洲蟑螂全身提取物的蛋白水解活性降低80%（1毫摩尔），抑制剂乙二胺四乙酸使平菇孢子的蛋白水解活性降低95%（1毫摩尔）。通过放射性变应原吸附试验抑制和免疫印迹法测定的变应原活性丧失与蛋白酶活性相关。因此，在变应原提取物的制备和处理过程中，应采用能使蛋白水解作用最小化的条件。
