Microplate assays with 96 wells were optimized to screen proteolytic activities in mite homogenates. Whole-mite extracts of Acarus siro, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Tyroborus lini, Carpoglyphus lactis, Lepidoglyphus destructor, and Dermatophagoides farinae exhibited non-specific proteolytic activity in buffers from pH 2 to 12, and three peaks of highest activity at pH 3, 5-6, and 10 were distinguished. The reducing agent Tris(2-carboxyethyl)phosphine hydrochloride decreased general proteolytic activity on azocasein at pH 5 and 6. The results obtained on two non-specific substrates, azocasein and azoalbumin, showed highly different ranks of the species at pH 5 and 6. Proteolytic activities toward N(α)-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride, N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide, N-Succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide, Benzyloxycarbonyl-L-arginine-L-arginyl 4-nitroanilide, and N-Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine 4-nitroanilide (MAAPMpNA) were highest at alkaline pH, but the activity toward MAAPMpNA was also high at pH 5 and 6. In contrast, N-Succinyl-L-alanyl-L-alanyl-L-phenylalanine 4-nitroanilide (AAPpNA) and L-arginyl 4-nitroanilide (ArgpNA) had the highest activity recorded at pH 6. The high activities observed on AAPpNA, ArgpNA, and MAAPMpNA at digestive pH suggest that enzymes present in these extracts could have the majority of proteolysis in the mite gut. Evidence of the presence of proteolytic activities on all tested substrates and in all the tested mite homogenates suggests that the proteolytic activities may contribute to allergenicity. Poor or undetected hydrolytic activities of mite extracts toward substrates for keratin and collagen at digestive pH underline the importance of ecological interactions between mites and microorganisms in the utilization of such substrates.

Crop Research Institute Praha Ruzyne Czechia

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