Catalase from larvae of the camel tick Hyalomma dromedarii

Abstract

Catalase plays a major role in protecting cells against toxic reactive oxygen species. Here, Catalase was purified from larvae of the camel tick Hyalomma dromedarii and designated TLCAT. It was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephacryl S-300 and CM-cellulose columns. Gel filtration and SDS-PAGE of the purified TLCAT indicated that the protein has a native molecular weight of 120 kDa and is most likely a homodimer with a subunit of approximately 60 kDa. The Km value of TLCAT is 12 mM H₂O₂ and displayed its optimum activity at pH 7.2. CaCl₂, MgCl₂, MnCl₂ and NiCl₂ increased the activity of TLCAT, while FeCl_2, CoCl₂, CuCl₂ and ZnCl₂ inhibited the activity of TLCAT. Sodium azide inhibited TLCAT competitively with a Ki value of 0.28 mM. The presence of TLCAT in cells may play a role in protecting H. dromedarii ticks against oxidative damage. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of untraditional methods to control them.

Keywords: BSA, bovine serum albumin; CAT, catalase; Camel tick; Catalase; Characterization; PAGE, polyacrylamide gel electrophoresis; Purification; ROS, reactive oxygen species; SOD, superoxide dismutase; TLCAT, tick larvae catalase; larvae.

Fig. 1

(a) A typical elution profile for the ammonium sulfate containing CAT activity fraction of the camel tick H. dromedarii larvae crude extract on DEAE-cellulose column (12 cm×2.4 cm i.d.). (b) A typical elution profile for the chromatography of the concentrated pooled DEAE-cellulose fractions containing TLCAT on Sephacryl S-300 column (142 cm×1.75 cm i.d.) (c) A typical elution profile for the concentrated pooled Sephacryl S-300 fractions containing TLCAT on CM-cellulose column (4 cm×1.6 cm i.d.).

Fig. 2

(a) Electrophoretic analysis of TLCAT on 7% native PAGE: (1) crude extract, (2) DEAE-cellulose fraction, (3) CM-cellulose fraction and (4) TLCAT activity. (b) Subunit molecular weight determination by electrophoretic analysis of TLCAT on 12% SDS-PAGE: (1) molecular weight marker proteins and (2) denatured purified TLCAT.

Fig. 3

(a) Lineweaver–Burk plot relating the reciprocal of the reaction velocity of the purified TLCAT to H₂O₂ concentration in mM. (b) Effect of pH on the purified TLCAT using 0.05 M potassium phosphate buffer, pH (5.7–8.0).

Fig. 4

(a) Inhibition of the purified TLCAT by varying concentrations of NaN₃. (b) Hill plot for inhibition of the purified TLCAT by varying concentrations of NaN₃.

Fig. 5

(a) Lineweaver–Burk plots showing the type of inhibition of purified TLCAT by NaN₃. (b) Determination of the inhibition constant (K_i) value for the inhibition of TLCAT by NaN₃.