Two-dimensional analysis of proteinase activity

A method was developed to separate proteinases in a complex mixture in two dimensions followed by activity detection using class specific substrates. Using this method, serine proteinase activity was evaluated in gut extracts from a stored-product pest, Plodia interpunctella. With the substrate n-α-benzoyl-l-arginine ρ-nitroanilide, three major groups of at least six trypsin-like activities were identified, consisting of proteinases with estimated molecular masses of 25–27, 40–41, and 289 kDa, and all with an acidic pI of 4.7–5.5. With the substrate, n-succinyl-ala-ala-pro-phenylalanine ρ-nitroanilide, two groups of at least five chymotrypsin-like activities were detected, with estimated molecular masses of 28 and 192 kDa and pI values ranging from 6.1 to 7.3. Using the 2-DE activity blot method, information was obtained on the relative number and physical properties of serine proteinases in a mixture of insect gut proteinases without prior fractionation.