Fig 5. Characterisation of LdNTR2.

(A) Purification of recombinant LdNTR2 from E. coli BL21(DE3)pLysS [pET15b-LdNTR2]. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, pooled fractions from Ni²⁺-affinity chromatography; lane 4, soluble protein following removal of histidine tag; and lane 5, pooled fractions from size-exclusion chromatography. MALDI analysis confirmed that the minor bands represent NTR2 degradation products. (B) Gel filtration profile of the LdNTR2. The inset shows a plot of elution volume against the log molecular mass (MW) of a standard protein mixture (black circles). The red circle represents the elution volume of NTR2. (C) Metabolism of nitroheterocyclic compounds by recombinant NTR2. Initial rates of metabolism were measured in assays containing 100 μM nitro-compound, 100 μM NADPH and 500 nM NTR2. Rates of metabolism with NADPH and NADH alone were 0.0124 ± 0.001 and 0.0084 ± 0.0006 μmol min^-1 mg^-1, respectively. Rates represent the mean ± SD of triplicate measurements. (D) Immunoblots of whole cell extracts (equivalent of 5×10⁶ parasites in each lane) from L. donovani mid-log promastigotes (L), metacyclic promastigotes (M) and axenic amastigotes (A) were probed with LdNTR2-specific polyclonal antiserum. Known amounts of purified recombinant LdNTR2 were loaded as standards for the quantification of the cellular levels of NTR2.