The fluorescence spectra were recorded in a PerkinElmer LS5S luminescence spectrometer. The intrinsic fluorescence spectrum was monitored from 300-400 nm upon excitation at 280 nm in a 0.5 cm path length cuvette. The concentration of the protein used was 0.1 mg/ml. 0.5 mg of bovine serum albumin per ml, in the presence or absence of 0.2 mg per ml poly(A) for 30 min. and the reaction was stopped by the addition of 100 mM EDTA (1 ml). The total volume of the reaction mixture was 10 ml. For kinetic studies, initially a stock of cold NTPs containing 0.008 mCi of 32 P labelled respective NTP was prepared and the substrate concentration was varied from 100-1000 mM for ATP and 10-80 mM for CTP, GTP, UTP and 2.5-20 mM for dATP. The reaction mixture was incubated with 2 mg of rNSs protein in the assay buffer. To determine the kinetic parameters of D159A mutant NSs, 10-70 mM ATP was used with 2 mg of mutant protein. Three independent sets of experiments were carried out to determine the kinetic parameters. Substrate hydrolysis was measured by Imagegauge software (Fujifilm) and the enzyme activity (v) was calculated using the formula given below: