The PD10 column is prepacked with Sephadex G-25 which works in the principle of size exclusion chromatography. The pores of Sephadex beads allow small molecular weight substances to pass through and prevents high molecular weight one from entering into the pores. Hence, the order of elution is first high molecular weight followed by low molecular weight as low molecular weight substances will take longer time to move. In this case, PD-10 column is used to transfer the protein to ammonium carbonate buffer in order to carry out trypsinolysis.
PD10 column, Eppendorf tubes, 50mM Ammonium Carbonate buffer pH 8.0, column stand
1. Column Preparation-
The sealed end at bottom of column was removed and storage solution was allowed to pass through. Then, the column was washed with 25 mL of double distilled water.
2. Column Equilibration-
Column was equilibrated with 25 mL of 50mM ammonium carbonate buffer.
3. Sample Application-
2 mL of sample were poured into the column and allowed to enter the column completely. Then additional ammonium carbonate buffer was added to carry out elution.
The position of hemocyanin in the column can be traced by its blue color. Eppendorf tubes were used to collect the protein at the bottom of column.
QUANTIFICATION OF HEMOCYANIN
The absorbance of hemocyanin in the solution is measured using Thermoscientific UV-Vis spectrometer at 280 nm and concentration is calculated using the Beer-Lamberts law as molar extinction coefficient of hemocyanin is known. (The molar extinction coefficient of hemocyanin is 1.5 cm-1 mg ml-1 at 1mg ml-1)
A = ?. l. c
A= Absorbance at 280 nm
?= Molar extinction coefficient of the protein
l= Path length of cuvette
c= concentration of protein
Molar extinction coefficient of a protein is the absorbance at 280 nm at a particular concentration. If the specific absorptivity or molar extinction coefficient of a protein is known, then using Beer-Lamberts law, the exact concentration of that particular protein can be calculated in the sample.
Thermoscientific UV-Vis spectrometer, 50 mM ammonium carbonate buffer (pH 8.0), Quartz cuvette, Double distilled water
I. The hemocyanin was diluted to 100 and 200 times (1:100 and 1:200 respectively) using the ammonium carbonate buffer.
II. The baseline of UV-Vis spectrometer was adjusted using ammonium carbonate buffer where the 3 mL of buffer were pipetted into each reference cuvette and sample cuvette.
III. The buffer is sample cuvette was replaced with the diluted sample and absorbance was taken at 280 nm. Measuring was done starting with low concentration sample.
IV. Finally, the concentration of hemocyanin in the sample was calculated using above Beer-Lamberts law.
TRYPSINISATION TO ISOLATE FUNCTIONAL UNIT OF HEMOCYANIN
Trypsin has an average molecular weight of 23.29kDa and works best at 370C with an optimum pH of 8.0. It is a serine protease which cleaves the protein at the carboxyl side of lysine and arginine specifically. Tryptinisation can be inhibited by freeze shock and by PMSF (Phenylmethanesulfonyl fluoride). To isolate functional unit from hemocyanin, limited trypsinsation is carried out in ammonium carbonate buffer pH 8.0 and incubating at 370C.
Trypsin(1mg/ml), incubator, PMSF, ice, hemocyanin in Ammonium carbonate buffer
1. Hemocyanin (32mg/ml) and trypsin(1mg/ml) were mixed in ratio of 300:1 respectively. That is, the reaction mixture contains 106?g of trypsin and 32000?g of hemocyanin for 1ml of hemocyanin.
2. The reaction mixture was incubated at 37°C in incubator for 3 hours.
3. After 3 hours the mixture was taken out. Then PMSF was added and freezed on ice to inhibit tryptic activity.
Dialysis is a process of diffusion where molecules move from higher concentration to low concentration. The dialysis tube has a specific molecular weight cutoff(MWCO). Molecules having MW of less than MWCO inside the dialysis tube will move out of the tube whereas molecules having MW of more than MWCO will be retained inside the tube. Here, the MWCO of the dialysis tube is 25kDa.
Dialysis tube(25kDa), FPLC Buffer A (0.05M Tris-HCl, 0.01M EDTA, 4M Urea, pH 8.9), Magnetic stirrer
About 5-6 cm of dialysis was cut using a sterile scissor and tube was kept in double distilled water for 24 hours at 4°C. Then the tube was washed with double distilled water till the water flow through the tube smoothly.
1. One end of the tube was tied with rubber band. Then 1ml of trypsinised hemocyanin was loaded into the dialysis tube. The other end was also tied with the help of rubber band.
2. Then the tube was kept in falcon tube containing Buffer A and was allowed to dialyze for about 5 hours on a magnetic stirrer.
3. Then the tube was taken out and dialyzed with fresh Buffer A and the same procedure was followed for 5 times.
4. Dialyzed hemocyanin was the taken for FPLC (Fast Protein Liquid Chromatography).
FAST PROTEIN LIQUID CHROMATOGRAPHY
Fast Protein Liquid Chromatography(FPLC) is a type of column chromatography which separates and purifies the proteins based on their charge. The column has two phases; mobile and stationary. The stationary phase can be solid, liquid or matrix. It contains negatively or positively charged beads to which the proteins bind for separation. To separate negatively charged proteins, anion exchange column is used where stationary phase is positively charged whereas cation exchange column contains negatively charged beads as stationary phase to separate positively charged proteins.
In this experiment, AKTA PRIME PLUS FPLC instrument was used where the Resource Q column was used to purify the functional unit of hemocyanin. Resource Q column was prepacked with Source 15Q (Quaternary Ammonium) which is positively charged and hence is a strong anion exchanger.
The net charge on the protein depends on the pH of the buffer used. If pH of buffer is less than pI(Isoelectric point) of the protein, the net charge on protein will be positive. If pH of buffer is more than pI, then net charge on protein will be negative. Isoelectric point of a protein is the pH at which net charge on protein is zero.
The pI of hemocyanin G type of Enteroctopus dofleinin, hemocyanin type 2 unit of Rapana venosa, ?C HiH of Helix lucorum and ?-C chain unit G hemocyanin of Helix pomatia are 5.60, 5.16, 5.2 and 5.48 respectively. As all the above three belongs to phylum Mollusk to which Pila globosa also belongs, the structural similarities between the hemocyanins also will be quite similar. Hence they also should have similar pI values. That is about 5.0 to 6.0. Protein in the buffer having pH more than this pI will provide protein a negative net charge. Hence, the pH of buffer is set to 8.9 where protein will have a strong negative net charge.
AKTA PRIME PLUS FPLC instrument, Resource Q column, Buffer A(0.05M Tris-Hcl, 0.01M EDTA, 4M Urea, pH 8.9), Buffer B(0.05M Tris-Hcl, 0.01M EDTA, 4M Urea, 0.8M NaCl, pH 8.9), Trypsinized hemocyanin in Buffer-A
Purification through FPLC has five steps are as follow-
Equilibration of column- The Resource Q column was equilibrated with 30ml of Buffer-A, that is 5 column volume.
Sample Application- 2ml of sample was injected via injection valve of the instrument in Load Mode. Air bubbles were avoided entering the column.
Wash I- 24ml of Buffer-A was used to wash the column to remove unbound or partially bound proteins.
Gradient Elution- Elution was carried out using a salt gradient from 0% to 100%. This gradient was established using Buffer-B which contains NaCl as salt. The chloride ion replaces the protein and proteins are eluted out.
Wash II- This step is also called as regeneration of column where 30ml of Buffer-A is used.
Pressure Limit 0.5MPa
Flow rate 1ml/min
Fraction Size 2ml
Equilibration Volume 30ml
Sample Injection Volume 2ml
Wash I Volume 24ml
Elution Volume 180ml
Wash II 30ml
Acetone precipitation is carried out in order to remove high amount of salt and other substances like urea from the solution so that they should not interfere in downstream studies like SDS-PAGE. Cold acetone is added to the protein solution where a protein precipitates which can be recovered via centrifugation.
As acetone precipitation denatures proteins, in order to remove salt from the solution, several other methods like dialysis, desalting etc. are carried out.
Cold acetone (stored at -20°C), refrigerator, Eppendorf tubes
i. 100µl of protein elution was pipetted into a fresh 1.5ml Eppendorf tube.
ii. Then 400µl of cold acetone was added to protein sample. The mixture was vortexed for about 1-2 minutes.
iii. The mixture was then kept in refrigerator for about 18 hours at -20°C.
iv. After 18 hours the mixture was centrifuged at 13,000 rpm for 10 minutes at 4°C.
v. The supernatant was discarded and pellet was air dried till acetone evaporated.
vi. The pellet was resolubilised with 1X reducing loading buffer for SDS-PAGE.
SDS-PAGE is Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis which was first described by Laemmli (1970). SDS denatures the secondary and tertiary structure of protein and provides an overall negative charge on protein. ?-mercaptoethanol breaks disulfide bonds in the protein. Because of this action charge of all proteins are same. Hence the proteins are separated in gel based on their molecular weight. Laemmli SDS-PAGE has discontinuous buffer system containing two separate gels-the upper stacking gel and bottom resolving gel. In stacking gel, the pore size is bigger because of acrylamide concentration as compared to resolving gel. As the pH is 6.8 in stacking gel order of movement in first chloride ion followed by protein and at last glycine. This ensures that proteins are in a straight line before entering resolving gel. When they enter the resolving gel pH increases to 8.8 and order of movement is first chloride ion followed by glycine and then protein. Here, the proteins get separated based on their molecular weight. Small proteins move through the resolving gel quickly as compared to large proteins.
Reagents- SDS, APS (Ammonium persulfate), TEMED (N, N, N, N-tetramethylethylenediamine), Acrylamide, Bis-acrylamide, Tris-base, ?-mercaptoethanol, Bromophenol blue, glycine, glycerol, methanol, Coomassie brilliant blue R-250 stain, glacial acetic acid, double distilled water
Equipment and glassware- Bio-Rad SDS-PAGE electrophoresis apparatus, pipettes, Eppendorf tubes, weighing balance, graduated cylinder, heater
SDS-PAGE reagents- 1.5M Tris-HCl pH 8.8, 0.5M Tris-HCl pH 6.8, 10% SDS, 10% APS, 1X reducing loading buffer (0.5mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 1% ?-mercaptoethanol, 0.1% BPB), electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)