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Objectives: To perform protein purification by ion exchange chromatography .

Principle : Ion exchange chromatography works on the basic principle that oppositely charged particles are attracted to each other .The stationary phase consists of fixed charges on a solid support .These charges can be either positive or negative .Hence , there are two types of ion exchangers i.e. cation and anion exchangers Cation exchanger possess negatively charged groups and attract positively charged molecules , e.g. Carboxymethyl cellulose or CM cellulose .Conversely , anion exchanger have positively charged groups that attract negatively charged molecules .e.g. , Diethylaminoethyl cellulose . Proteins are complex ampholytes i.e. they have both positive and negative charges and can be separated from a mixture of compounds on the basis of net positive and negative charge that they can carry .Iso -electric point of a protein (pI) is that pH at which its net charge is zero (i.e. the number of positive and negative charges are equal ) . Therefore , Proteins will have either a net positive or negative charge depending on the pH of the solution and thus , it is possible to use either an anion exchanger or a cation exchanger . In ion exchange chromatography , a solution containing protein of interest is applied to the ion exchanger .Protein binding to the ion exchanger depends on the net charge of that protein at that particular pH and on the ionic strength of the mobile phase .Bound protein is then eluted out from the stationary phase by increasing the concentration of counter ions or by changing pH , which alters the charge on that protein .Weakly charged ions are displaced from the stationary phase with lower concentration of counter ions than highly charged proteins .This results in the separation of the protein based on its net charge . Extent of purification of a protein ( e.g. ; enzyme ) can be determined by computing its specific activity .Specific activity is the ratio of enzyme activity to mass of protein in the sample , usually expressed as units of activity per milligram of protein ( U/mg). As the enzyme is purified ( through a number of steps ) other proteins in the mixture are eliminated while most of the enzyme activity is retained , this results in an increase in the specific activity of the enzyme .Hence , by determining specific activity before and after purification , one can determine the fold purification and yield of the enzyme . The extraction and purification of lysozyme will be carried from chicken egg white by ion exchange chromatography . The pI of lysozyme is 10.5 and it carries a net positive charge at the pH below 10.5 . Hence , at pH 7.0 , it binds to negatively charged column or a cation exchanger .Wash buffer ( pH 9.0 ) will then be used to remove the protein that have pI less than or equals to 9.0 .Following which ,

Lysozyme will be eluted out by increasing concentration of cations . Cations compete with positively charged groups of lysozyme for bindibg sites on the column , resulting in elution of lysozyme Lysozyme activity and protein concentration of the crude and purified sample are as follows ; Enzyme activity of the lysozyme is determined using the bacterium Micrococcus luteus .A suspension of intact bacteria is cloudy ,absorbing light strongly at 405 nm .As lysozyme breaks down the cell walls , bacterial membranes break open due to osmotic shock , the breakdown product dissolve and the suspension becomes clearer .Thus as lysozyme acts , the absorbance of the substrate suspension decreases . Hence , one unit of lysozyme is defined as the amount of lysozyme that will produce a decrease in absorbance at 450 nm of 0.001 absorbance units/minute . Protein concentration of lysozyme is estimated by measuring the UV absorbance at 260 nm and 280 nm This value will be used to calculate specific activity and yield of lysozyme .Specific activity of lysozyme before and after purification will be estimated by comparing its activity with that of a standard lysozyme whose specific activity is known . Materials required : CM cellulose 10 X Equilibration buffer ( pH 7.0) 10X wash buffer ( pH 9.0 ) 5X Elution buffer Neutralizing solution 5M Sodium chloride 0.5 M phosphate buffer (pH 7.0 ) Tube for mixing Micrococcus luteus Lysozyme standard Column Equipment required Centrifuge , Spectrophotometer. Reagents: Chicken egg Distilled water

Glasswares Beakers , test tubes . Other requirements : Column stand , micropipette , Tips , quartz cuvette.

Procedure : Purification of lysozyme using CM cellulose ; 1. Wash the empty column with hot water ( 90 degree Celsius ) 2. Fix the column to the stand .Remove the top cap of the column and pack the column with 2.5 ml of CM cellulose . 3. Remove the bottom cap and equilibrate the column with 50 ml of 1X equilibration buffer . 4. Break an egg and collect the egg white separately . 5. To 6 ml of egg white , add an equal volume of distilled water .Mix in the tube for 10 min to get the homogenous solution . 6. Adjust the pH of the egg white to 7.0 , by slowly adding neutralizing solution . 7. Centrifuge the egg white solution at 6000 rpm for 10 minutes .Collect the supernatant . 8. Save 0.5 ml of the supernatant for measurement of lysozyme activity ,label this as crude sample . 9. Load rest of the supernatant to equilibrated CM cellulose column . 10. Replace the top and bottom caps of the column and incubate for 1 hour at room temperature with intermittent mixing . 11. After an hour , allow the column material to settle .Slowly pipette out or decant the supernatant without disturbing the column . 12. Wash the column with approximately 30 40 ml of 1X Wash buffer . 13. Elute lysozyme from the column using 15 ml of 1X elution buffer . 14. Start collecting the eluate in test tubes a 2 ml fractions .Monitor OD at A 280 and pool the fractions that show A 280 0.5 and above .Label this as eluate . 15. Wash the column with 10 ml NaCl .Replace the top and bottom caps .Store at 4 degree Celsius , for next use .

Estimation of protein concentration : Crude sample: 1.Dilute the crude sample 20 times with PB .( i.e. 0.2 ml of egg white made upto 4 ml .). 2. Zero the spectrophotometer against phosphate buffer blank .

3. Measure the absorbance at 260 and 280 nm. 4. Use the following formula to calculate concentration of protein in crude sample : Protein concentration = [ ( 1.55 * absorbance at 280 nm) - (0.77 * absorbance at 260 nm )] * dilution factor ( dilution factor is 20 times ) =------------- mg / ml , Eluate : 1. Dilute the eluate 10 times with PB ( i.e. 0.3 ml made upto 3 ml ). 2. Zero the spectrophotometer against phosphate buffer blank. 3. Measure the absorbance at 280 nm. 4. Use the following formula to calculate concentration of proteins in eluate . Protein concentration =absorbance at 280 nm * dilution factor =------- mg/ml 2.55 where , dilution factor = 10 2.55 is the extinction coefficient of lysozyme i.e. , absorbance of 1 mg/ ml of lysozyme is 2.55 .

Estimation of lysozyme activity : 1.Dilute required amount of reconstituted standard lysozyme 1:3 i.e. ( 0.3 ml of standard lysozyme + 0.6 ml of phosphate buffer ) to bring down the concentration to 0.33 mg /ml ( from 1 mg /ml to 0.33 mg / ml ) . 2.Dilute the crude sample and eluted sample to 0.33 mg/ml based on the protein concentration estimated .For example , if the concentration is 1 mg /ml , dilute it three times with phosphate buffer to bring down the concentration to 0.33 mg /ml . 3. Zero the spectrophotometer against phosphate buffer blank. 4. Dilute required amount of Mlu in phosphate buffer such that absorbance 450 nm is between 0.5 and 0.7 . 5. Take 3 ml of diluted Mlu substrate in a cuvette and measure the absorbance at A 450 against phosphate buffer blank , this is A 450 at 0 second. 6. To the substrate , add 50 l of standard enzyme and note the time . 7.Mix the contents of the cuvette for 15 seconds .

8.Measure the absorbance exactly after 60 seconds of addition of lysozyme . 9.Repeat steps 5 to 8 for the crude and eluted samples . 10. Note down the readings as I following Time Standard 0 sec 60 sec Delta A 450 OD readings of lysozyme activity Estimation of the specific activity of lysozyme : Specific activity of lysozyme in crude and eluted samples is calculated by comparing the OD readings of the sample with that of standard lysozyme , whose specific activity is known . Activity in u / mg = Delta A 450 / min . of the test * activity of the standard Delta A 450 / min of the standard . Where Delta A 450 is the difference in A 450 at 0 and 60 seconds . ( Activity of standard is 48, 000 u /mg . ) Estimation of fold purification : Fold purification = Specific activity of eluted sample / specific activity of crude sample This is a measure of the efficiency of purification of lysozyme using ion exchange chromatography . Estimation of yield . Yield = protein concentration of eluate * total volume of eluate = ------------- mg Result : C1 Total volume Crude C2 Protein concentration ( mg /ml ) C3 Total protein ( in mg ) C1* C2 C4 Specific activity ( u /mg ) C5 Total yield( U) C3 * C4 A 450 Crude

Eluate

sample Eluate For myself : 1. The duration of the experiment is for 5 hours . 2. All materials required should be stored at 4 degree Celsius . 3.We need to handle the neutralizing solutions carefully as it is corrosive . 4.We need to store the micro- coccus