Isolating or purifying a biomolecule from its natural surroundings is often necessary for biochemists because there are often other molecules present which interfere. Separation of biomolecules from each other is based upon exploiting some difference between them. We have seen how the physicochemical property of a molecule’s charge can be used by electrophoresis, in this experiment you adopt a morepreparative approach than purely analytical: we are going to prepare a good amount of enzyme for subsequent study of the reaction it catalyses using charge.Ion exchange is the exchange of ions of like sign between a solution and an insoluble solid body in contact with it.

There are thus two types of ion exchangers:

Cation exchangers in which the solid body carries negative charges and positive charged soluble cations bind to it.Anion exchangers in which the reverse is the case.Within each of the above categories, the ion exchangers differ according to the acidity, or basicity, of theimmobile charge. The following shows the ion exchangers most often used in protein purification. (Note:other types of ion exchange resins such as "Dowex" and "Amberlite" are used in the extraction ofmicromolecules.Anion ExchangersDiethylaminoethyl (DEAE) weak base -O-CH2CH2N+H (C2 H5)2Quaternary amino-ethyl (QAE) strong base -O-CH2CH2 N+(C2 H5 )2|CH2 CH (OH) CH3Cation ExchangersCarboxymethyl (CM) weak acid -O-CH2 COO-Sulphonyl (SP) strong acid -CH2 CH2 CH2 SO3 -In this experiment you will be using the cation exchanger CM-cellulose to purify lysozyme from eggwhite. Lysozyme has a pI of 11. (i.e. it carries a net charge of zero when in a solution at pH 11). Thisis higher than most other proteins. It is also stable at pH 10. This means that if the pH of egg white israised to 9.5, of all the proteins present, lysozyme alone will still carry a positive charge. (A minorconstituent of egg-white, avidine, behaves similarly). If one were to use an anion exchanger at pH 10, thelysozyme would not bind whereas all other proteins would bind. In order to ensure that all of the anionicproteins would bind, a large quantity of anion exchanger would be required. It is therefore moreeconomical (and, all other things being equal, therefore better) to use a cation exchanger which will bindonly the lysozyme and leave the other 90% of the egg-white constituents in solution.

Lysozyme purification by ion exchange55PURIFICATION PROCEDUREThe egg white was filtered through muslin and diluted five fold with 50 mM Na2CO3/NaOH buffer pH9.5. Collect and store a sample of this on ice for analyses later.1. To 5 ml of the diluted egg white add 5 g of CM-cellulose equilibrated in 50 mM Na2CO3/NaOHbuffer pH 9.5; adjust the pH, using HCl or NaOH as necessary, with a calibrated pH meter and stirgently for 3 mins or place in stoppered tube and shake.2. Centrifuge the suspension to give a clear supernatant, for approximately 3 minutes.3. Decant the supernatant; record the volume of this 1st supernatant and store it suitably labelled on ice.4. Re-suspend the CM pellet in 5 ml pH 9.5 buffer, check and adjust the pH if necessary and stir orshake gently for 3 mins.5. Centrifuge the suspension as before; record the volume of this 2nd supernatant and store it suitablylabelled on ice.6. Re-suspend the filter cake in 5 ml pH 10.5 buffer, check and adjust the pH if necessary and stir orshake gently for 5 mins.7. Centrifuge the suspension as before, record the volume of this 3rd supernatant, pH 10.5, (whichshould contain much of the lysozyme) and store it suitably labelled on ice. Wash the pellet of CMcellulose into the container provided so that it may be recycled.You now have a sample of the original diluted egg white, the first two supernatants at pH 9.5, and thefinal supernatant at pH 10.5.ASSAY OF LYSOZYME ACTIVITYLysozyme dissolves certain bacteria by cleaving a glycosidic bond in the peptidoglycan component oftheir cell walls, enabling osmotic pressure to burst the cells. A suspension of susceptible bacterial cells inthe light path of a spectrophotometer will scatter the incident light away from the detector and thus reducethe light transmittance. As lysozyme dissolves the bacteria the light scattering effect is reduced and thetransmittance of light increases.Substrate: Micrococcus lysodiekticus 25 mg/100 ml 0.1 M phosphate buffer pH 6.2Place 1.0 ml substrate in a cuvette in a spectrophotometer zeroed with air reading at 540 nm. Theabsorbance should read between 0.4 and 0.8 units. Add 0.1 ml of your sample and start timing. Quicklymix the cuvette thoroughly (by inverting several times) and place in light path. Take OD readings at 10-second intervals for two minutes. If the OD drops too fast to measure, dilute a portion of your sample bya known amount and try again.SUGGESTED DILUTIONS:Egg white solution: 10-fold, 25-fold, and 50-fold1st supernatant: no dilution2nd supernatant: no dilutionpH 10.5 supernatant: no dilution, 10-foldNote: A ten-fold dilution means that the same amount of stuff is now in ten times the volume, so tomake a ten-fold dilution take 1 ml of original sample and add 9 ml of diluent to make a final volumeof 10 mls.ResultsPlot the 4 results on one graph of OD vs. time (10 MARKS);Results to be handed in at your next laboratory session1. Determine the initial (steady) rates. Take as units of activity the drop in OD 540 nm per minute. Youwill have a value for each sample. Multiplying by the appropriate factors, determine the total activitypresent in all fractions. (20 MARKS)

Lysozyme purification by ion exchange562. How much of the activity that you started with did you recover? Assuming that you recovered lessthan 100%, where is the remaining activity, and how might this be recovered? Assuming that youapparently recovered more activity than you originally started with, how could one explain this?