Biochemical and Immunochemical Properties of HPLC Peak 2, an Ion-Exchange Fraction of Common Cattle Grub (Diptera: Oestridae)

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Abstract
Soluble proteins extracted from first instar common cattle grub, Hypoderma lineatum (Villers), can be resolved into four major proteolytic fractions by HPLC ionexchange chromatography. Hypodermin A, hypodermin B, and collagenase are serine proteases contained in three of the four fractions and have been described previously. HPLC peak 2 (P2), another fraction of first instar H. lineatum, was prepared and compared with the three other protease fractions. P2 exhibited tryptic esterase activity (as indicated by hydrolysis of amide and ester substrates of trypsin) and no chymotryptic esterase activity. Although tryptic activity within P2 was inhibited by both soybean and bovine pancreatic trypsin inhibitors, chicken ovomucoid had little inhibitory effect. Ethylenediaminetetraacetic acid (EDTA) and N-α-tosylphenylalanine chloromethyl ketone (TPCK, a chymotrypsin inhibitor) were without effect on P2 tryptic esterase activity, while the trypsin inhibitors benzamidine and N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) were slightly inhibitory. Tryptic esterase activity of P2 was relatively stable at temperatures from 20 to 50°C and had pH optimum of 7.0-7.8. Results indicated that hypodermin B and P2 protease activities were more closely related to one another than they were to hypodermin A. lmmunochemical cross-reactivity suggested antigenic similarities between hypodermin B and P2, yet hypodermin A was found to be antigenically distinct.

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