Fig. 8

Histidine residues are essential for nuclease activity of hPLSCR1. a Schematic showing the position of five histidine residues in hPLSCR1. F1-F6 and R1-R6 were the forward primers and reverse primers used for generation of mutations respectively. A detailed methodology for generation of point mutants is described in 'Methods' section. b Silver stained SDS-PAGE gel showing 20 pmol of Mut-hPLSCR1 along with WT-hPLSCR1 purified to homogeneity. Circular Dichroism studies for WT-hPLSCR1 (c) and Mut-hPLSCR1 (d) in the presence and absence of 3 mM MgCl₂ were shown. e Nuclease assay for Mut-hPLSCR1 (gel assay): Gel assay was performed for Mut-hPLSCR1 and WT-hPLSCR1 as described in ‘Methods’ section and visualized on a 1 % agarose gel. f Dose dependence of Mut-hPLSCR1 by gel assay: Nuclease assay was performed at increasing concentration of Mut-hPLSCR1 (20 pmol, 40 pmol, 60 pmol) and visualized on a 1 % agarose gel. g Dose dependence studies for Mut-hPLSCR1 by Kunitz assay: Kunitz assay was performed to quantify the nuclease activity of increasing concentrations of Mut-hPLSCR1 (20 pmol, 40 pmol, 60 pmol) (grey bars) and compared with the dose dependent nuclease activity of WT-hPLSCR1 (white bars). ** shows statistical significance at p < 0.005; ns- not significant. Experiments were repeated at least three independent times and the error bars denote standard deviation