**Abbreviations**

322 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 8.** FTIR analysis showed peak around 1646.936 in *P. aeruginosa* 2453 whereas, others showed

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

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

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

peak around 1642.269, 1639.446, 1637.873 for *P. aeruginosa* 741, 1942, 1934 respectively.

**4. Discussion** 

spectrometer.

molecular weight of Azurin.

MALDI-Matrix-Assisted Laser Desorption/Ionization, SDS-PAGE-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, FTIR- Fourier Transform Infrared Spectroscopy, CuSO4 – copper sulphate, KNO3 **–** Potassium nitrate, MTT -3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium, PI-Propidium Iodide, DMSO-dimethylsulfoxide, MALDI-ToF-Matrix-Assisted Laser Desorption/Ionization-Time of Flight, MTCC- Microbial Type Culture Collection center, CM-carboxymethyl, DEAE-Diethylaminoethyl Cellulose
