PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY INVOL

ESTIMATION OF ENZYME (LYSOZYME)ACTIVITY

ESTIMATION OF ENZYME (LYSOZYME)ACTIVITY (contd.)

Estimation of Yield

• Note down the readings as in Table 2: of Phosphate buffer to bring down the concentration to 0.25 mg/ml (from 1 mg/ml to 0.25 mg/ml)
• Dilute the crude sample and eluate to 0.25 mg/ml based on the protein concentration estimated. e.g.
• If the concentration of lysozyme is 1 mg/ml; dilute it four times with phosphate buffer to bring down the concentration to 0.25 mg/ml.

• Take a loopful of culture from the Micrococcus luteus (Mlu) slant into a test tube containing 5 ml of 0.1M
• Phosphate Buffer, pH 7.0 such that OD450 is between 0.5 to 0.7.
• Dilute required amount of standard lysozyme 1:4 i.e. (0.25 ml of standard lysozyme + 0.75 ml.

Yield = Protein concentration of eluate x total volume of eluate

= -------------------- mg

• AFTER AN HOUR ALLOW THE COLUMN MATERIAL TO SETTLE. SLOWLY PIPETTE OUT THE SUPERNATANT WITHOUT DISTURBING THE COLUMN.
• WASH THE COLUMN WITH 30-40 ml OF 1X WASH BUFFER.
• ELUTE LYSOZYME FROM COLUMN USING 15 ml OF 1X ELUTION BUFFER

MATERIALS REQUIRED

PRINCIPLE

• CHICKEN EGG WHITE
• DISTILLED WATER
• GeNei ION EXCHANGE KIT
• COLUMN STAND
• CENTRIFUGE
• SPECTROPHOTOMETER
• MICROPIPETTE
• TIPS
• QUARTZ CUVETTE
• BEAKERS
• 10ML MEASURING CYLINDER

Protein concentration of Crude sample

FORMULA TO CALCULATE CONCENTRATION OF PROTEIN IN CRUDE SAMPLE:

= [(1.55 X A280)-(0.77XA260)] X DILUTION FACTOR. (DILUTION FACTOR=20)

= 23 mg/ml

• CENTRIFUGE THE EGG WHITE SOLUTION AT 6000 rpm FOR 10 min.
• SAVE 0.5ml OF SUPERNATANT FOR MEASUREMENT OF LYSOZYME ACTIVITY, LABEL THIS AS CRUDE SAMPLE.
• LOAD REST OF THE SUPERNATANT TO EUILIBERATED CM-CELLULOSE COLUMN.
• REPLACE THE TOP AND BOTTOM CAPS OF THE COLUMN AND INCUBATE FOR 1 HOUR AT ROOM TEMPERATURE WITH INTERMITTENT MIXING.

• ION EXCHANGE CHROMATOGRAPHY WORKS ON THE BASIC PRINCIPLE THAT OPPOSITELY CHARGED PARTICLES ARE ATTRACTED TO EACH OTHER

STATIONARY PHASE CONSISTS OF FIXED CHARGES ON A SOLID SUPPORT.

• WE USED CATION EXCHANGER WHICH POSSESS NEGATIVELY CHARGED GROUPS AND ATTRACT POSITIVELY CHARGED MOLECULES. LIKE CARBOXYMETHYL –CELLULOSE OR cm-CELLOLOUSE.

INTERPRETATION

Estimation of Specific activity of Lysozyme:

• The lysozyme was effectively purified from chicken egg white proteins in a single step with reasonable yield and
• purity. The protein concentration was calculated and lysozyme activity was estimated by Micrococcus luteus assay.

• Specific activity refers to the “purity” of the sample with respect to the enzyme of interest and the total protein before and after the purification process.
• Specific activity is defined as the units of enzyme activity per mg total protein.
• Specific activity of lysozyme in crude and eluted samples is calculated by comparing the OD readings of the samples with that of standard lysozyme, whose specific activity is known.

PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY INVOLVING THE FOLLOWING EXPERIMENTS:

ESTIMATION OF ENZYME (LYSOZYME)ACTIVITY (contd.)

• BREAK AN EGG. COLLECT THE EGG WHITE SEPARATELY.
• TO 6ml OF EGG WHITE, ADD AN EQUAL VOLUME OF DISTILLED WATER.
• MIX IN THE MIXING TUBE PROVIDED FOR 10 MINUTES TO GET A HOMOGENOUS SOLUTION.
• ADJUST THE pH OF EGG WHITE TO 7.0 BY SLOWLY ADDING NEUTRALIZING SOLUTION.

ESTIMATION OF PROTEIN CONCENTRATION

• Zero the spectrophotometer against 0.1M Phosphate Buffer Blank.
• Take 1 ml of Mlu culture in a cuvette and measure the OD450 against phosphate buffer blank. This is OD at ‘0’second.

• DILUTE THE CRUDE SAMPLE 20 TIMES WITH PHOSPHATE BUFFER (0.2 ml OF EGG WHITE MADE UPTO 4 ml)
• ZERO THE SPECTROPHOTOMETRE AGAINST PHOSPHATE BUFFER BLANK.
• MEASURE THE ABSORBANCE AT A 260 AND A280

• PURIFICATION OF LYSOZYME USING CM-CELLULOSE
• ESTIMATION OF ENZYME ACTIVITY
• ESTIMATION OF PROTEIN CONCENTRATION

• START COLLECTING THE ELUATE IN TEST TUBES AS 2 ml FRACTIONS.
• MONITOR OD AT A 280 LABEL THIS AS ELUATE.

ESTIMATION OF ENZYME (LYSOZYME)ACTIVITY (contd.)

Observation and Result:

Calculate and tabulate fold purification of lysozyme

C

Fraction

B

A

D

E

• To the substrate, add 100 μl of standard lysozyme (0.25 mg/ml) and note the time.
• Mix the contents of the cuvette for 15 seconds.
• Measure the absorbance exactly after 60 seconds of addition of lysozyme.
• Repeat the steps 5-8 for crude and eluted samples.

TOTAL YIELD (C X D)

TOTAL VOLUME

LYSOZYME SPECIFIC ACTIVITY (u/mg)

TOTAL PROTEIN (mg) A xB

PROTEIN CONC.(mg/ml)

Estimation of Fold Purification:

CRUDE EXTRACT

ELUATE

Specific activity of eluted sample

Fold Purification = -------------------------------------

Specific activity of crude sample

This is a measure of efficiency of purification of lysozyme using ion exchange chromatography.

Estimation of Specific activity of Lysozyme(Cont.)

A450/min. of the test x activity of the standard

Activity in U/mg = ------------------------------------

A450/min. of the standard

Where A450 is the difference in A450 between 0 sec and 60 sec

Activity of standard lysozyme is 1,00,000 U/mg

PROCEDURE

CALCULATIONS

PURIFICATION OF LYSOZYME USING CM-CELLULOSE

A450

Time

Standard

Eluate

0 seconds

PROTEIN PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY

60 seconds

Δ A450

• WASH THE EMPTY COLUMN WITH HOT WATER
• FIX THE COLUMN TO THE STAND.
• REMOVE THE TOP CAP OF THE COLUMN AND PACK THE COLUMN WITH 2.5ml OF CM-Cellulose
• REMOVE THE BOTTOM CAP AND EQUILIBRATE THE COLUMN WITH 50 ml OF 1X EQUILIBRATION BUFFER.