*2.6.5. Purification profile of Azurin ((synthesized from P. aeruginosa MTCC2453) by SDS-PAGE*

Five ml of 12% resolving gel contains 1ml distilled water, 30% acryl amide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS and 0.002µl TEMED for polymerization was casted in the glass slab without any bubbles and kept it for 10-15 minutes. After polymerization of the resolving gel, 3ml of stacking gel (4%) were loaded over the resolving gel which contains 0.68 ml distilled water, 30% acryl amide, 1M Tris (pH 6.8) 10% SDS,10% APS, and 0.001ml TEMED. After casting the gel, proteins purified from different chromatography were loaded with bromophenol (molecular weight marker dye) at different lanes for profiling the protein purification process.

Glass slab gel were kept in the electrophoresis tank with tank buffer (196 mM glycine, 0.1%SDS, 50mM Tris-Hcl pH 8.3 made by diluting a 10x stock solution). This setup was connected with power pack initially in 80mV to 100 mV. After running the gel up to its anode end, was removed and stained with 0.2% coomassie brilliant blue for overnight. Destained with destaining solution (45: 45: 10 – methanol: water: acetic acid) which destains the comassie blue until it reveals the bands. The bands (figure 4) were observed under UV transilluminator (Biorad, PA, USA) [16].

## *2.6.6. FTIR analysis*

314 Chromatography – The Most Versatile Method of Chemical Analysis

minutes to remove all unattached proteins [11-13].

*2.6.2. Purification of Azurin on gel-filtration chromatography* 

pH attains its buffer pH [11-13].

[12-14].

*2.6.2.1. Chromatography on Sephadex G-25* 

*2.6.2.2. Chromatography on Sephadex G-75* 

collected at 1 ml/6minutes flow rate [12-14].

*2.6.3. Purification of Azurin on ion – Exchange (anionic) chromatography* 

*2.6.3.1. Chromatography on CM cellulose* 

buffer pH 4.65 [12-14].

dialysate and stirred for 20-30 min at 40C.The suspension was centrifuged at 10,000g for 15 min. Azurin does not adsorb in the gel remains in supernatant but most of the unwanted proteins like yellow flavo proteins are removed. Supernatant was collected. DEAE cellulose precipitate was resuspended in the same buffer and again centrifuged at 10,000g for 15

The supernatant after DEAE treatment was saturated to 100% (766g/l) with (NH4)2SO4 at 40C for overnight for precipitation. After saturation, precipitates are mixed gently and kept for centrifugation at 10000g for 10 min. supernatant was collected for dialysis at 40C for overnight with gentle stirring with the same before. Dialysis was continued till the solution

Sephadex G-25 beads were equilibrated in the 0.02M potassium phosphate buffer pH 7 [Parr S R et al 1976] for overnight, and tightly packed in 3cm x 25cm length glass column without any bubbles. The column was initially washed with 0.02M potassium phosphate buffer for twenty volumes of the gel packed. The Flow rate was adjusted to one minute per ml. slowly the dialysate (after DEAE treatment) was added with the eluent buffer 0.02M potassium phosphate buffer pH 7 on the column. Thirty fractions were collected at one minute interval

Sephadex G-75 beads in powder form are equilibrated in 0.01M Tris/Hcl buffer pH 7.5 for overnight. After equilibration the beads were tightly packed in a 3cm x 45cm glass column. The column was washed with same equilibrating buffer for fifty volumes of the column value. After washing with buffer one ml of the sample (fraction (a) collected from the G-25) were passaged and eluted with the same equilibrating buffer. Seventy five fractions were

The CM cellulose beads from Sigma–Aldrich (Kolkata, India) were equilibrated for overnight in the ammonium acetate buffer pH 3.9 adjusting the pH by 0.05M acetic acid with 2M NH3.After swelling, the beads were packed in a 5cm x 15cm glass column and washed for ten times of the column volume. Gently one ml of the sample added (Fraction (e) collected from G-75) over the top of the column and left it for 5-10 minutes to bind the protein inside the beads. After 10 minutes the column was eluted with ammonium acetate

Infrared spectroscopy experiments were performed using a Nexus 870 (Thermo Nicolet Corporation, Madison, USA) spectrometer equipped with a potassium bromide (KBr) beam splitter and DTGS (deuterated triglycine sulfate) detector in the range of 3,000-4000 cm−1. We recorded 32 scans per spectrum at a 2 cm−1 resolution for 100 µl of azurin liquid samples in 0.02 M PBS buffer (pH 7.0). We kept the same buffer as a background medium and performed all measurements at room temperature. We corrected spectra for the moisture

and carbon dioxide in the optical path. The curves were deconvoulted and imported into Omnic's peak fit software (Thermo scientific, Illinois, USA) and a Gaussian curve fitting performed [17].

Purification of Azurin from *Pseudomonas Aeuroginosa* 317

**Figure 2.** Quantification of azurin synthesis by different strains of *P. aeruginosa* MTCC 741, 1934, 1942 and 2453 and impact of CuSO4 and KNO3: 1-5 µg/ml range of CuSO4 concentration with 0.004-0.02 µg/ml of KNO3 was added in the culture medium to study the impact of azurin synthesis.

> *P. aeruginosa* MTCC 741

*P. aeruginosa* MTCC 2453 *P. aeruginosa* MTCC 1942

MTCC 1934

Total dry cell yield in g/l medium 1650 1780 **156 0** 1590

treatment in g/l medium 560 610 **<sup>460</sup>**<sup>490</sup>

treatment g/l. 440 485 **315** <sup>384</sup>

treatment g/l. 320 350 **295** <sup>302</sup>

dry bacteria. (CM cellulose) 2.9 2.4 **3.95** 2.6

**Table 1.** Azurin yield from different strains (*P. aeruginosa* MTCC 741, 1934, 1942 and 2453) in addition

DEAE and G-25 are gel filtration columns which remove positively and negatively charged proteins respectively. The unwanted flavo proteins and positively charged proteins were removed during DEAE chromatography. The collected fractions from G-25 were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin and other proteins more than 5 kDa were eluted immediately after void volume is plotted as

(NH4)2SO4 precipitation (g/l) 1520/1410 1460/1550 **1105/1250** 1205/1300

Purification step *P. aeruginosa*

of 5 µg/ ml CuSO4 and 0.02 µg/ml KNO3 in the culture medium

**3.3. Chromatography methods for azurin Purification** 

Protein concentration after 45/95 %

Protein concentration after DEAE

Protein concentration after G-25

Protein concentration after G-75

Total Azurin synthesis in mg/g

graph (Figure 3.).
