**4. Discussion**

Azurin production from *P. aeruginosa* MTCC 2453 was enhanced when 5µg/ml of copper sulphate and the potassium nitrate (0.02µg/ml) was added. At each step of the purification process protease inhibitor was added to the protein sample for inhibition of protein lyses. During dialysis most of the lower (approx) proteins up to 3 kDa molecular weight are pierced out. The retained proteins (above 3 kDa molecular weight) were washed in sephadex G-25 which serves as a desalting column and also has 3-5 kDa molecular weight fractionation range. High molecular weight more than 5 kDa proteins were eluted immediately after void volume which was revealed in SDS-PAGE and MALDI-ToF spectrometer.

The fraction collected from G-25 containing only more than 5 kDa proteins were passed through on G-75 which has 5-80 kDa fractionation range. The higher proteins above 80 kDa molecular weight elute after void volume; the remaining proteins between 6-70 kDa were bounded within the beads later eluted by the buffer. The fraction which showed 14 kDa molecular weight by analyzing in MALDI-ToF spectrometer for all the fractions (MALDI-ToF results not shown for all fractions which showed peak) were collected and again purified in CM cellulose chromatography. The fraction which showed peak in CM cellulose was again observed in MALDI-ToF spectrometer to confirm the presence of 14 kDa molecular weight of Azurin.

Our idea of adding copper in the culture medium was not only for the enhanced azurin synthesis, but to reveal the differences of azurin's stability in the secondary structure for all *P. aeruginosa* strains. The FTIR investigation showed azurin has C=O (protein backbone) stretching, which is the unique nature of the amide I band. The presence of the amide band at 1650 cm-1 signifies the α-helix secondary structure of azurin. The significant shift among four strains synthesized azurin implies that there was a difference in their secondary structure which may be due to their physiological or genetic variations among strains. The impact of the differences in the secondary structure of azurin synthesized from all four strains tested, were also reflected in the apoptosis generation of all strains.
