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. 2023 Mar 8;21(1):28.
doi: 10.1186/s43141-023-00486-w.

Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii

Hassan M M Masoud  1   2 , Mohamed S Helmy  3   4 , Doaa A Darwish  3   4 , Mahmoud A Ibrahim  3   4
Affiliations

Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii

Hassan M M Masoud et al. J Genet Eng Biotechnol. .

Abstract

Background: Glutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione.

Results: A GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg-1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver-Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg-1. TLGST exhibited its optimal activity at pH 7.9. Co2+, Ni2+ and Mn2+ increased the activity of TLGST while Ca2+, Cu2+, Fe2+ and Zn2+ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM.

Conclusions: These findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations.

Keywords: Camel tick; Characterization; Glutathione-S-transferase; Hyalomma dromedarii; Purification.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
a The chromatographic pattern of 40–80% ammonium sulfate fraction of tick larvae crude extract on GSH-Sepharose column (8 × 1.4 cm) previously equilibrated and washed with 0.02 M Na-phosphate buffer pH 7.0. b The chromatography of tick larvae TLGST on Sephacryl S-300 column (142 cm × 2.4 cm) formerly equilibrated with 0.02 M Na-phosphate buffer pH 7.0
Fig. 2
Fig. 2
a Analysis of TLGST on 12% SDS PAGE; (1) molecular weight marker proteins, (2) TLGST. b Analysis of TLGST on isoelectric focusing PAGE: (1) pI marker proteins, (2) TLGST
Fig. 3
Fig. 3
a The pattern of TLGST pH profile utilizing 0.1 M potassium phosphate (5.7–8.0) and Tris–HCl (8.0–9.2). b Lineweaver–Burk plot of TLGST reaction speed in response to CDNB concentrations
Fig. 4
Fig. 4
a TLGST Inhibition with pCMB various concentrations. b Hill plot of TLGST inhibition with pCMB. c Lineweaver–Burk plots showing TLGST inhibition type with pCMB. d TLGST inhibition constant (Ki) value for pCMB
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