FCH 530 Homework 3 Your assignment will not be accepted if it is not legible. Show your work !.

You are asked by your ad"isor to purify a protein from Escherichia coli cells. You know that your protein is intracellular #in the cytosol$ and that it has a p% of !0. &escribes the steps and techni'ues you might use to purify this protein. (i"en the following data) *mmonium 0 !0 Sulfate #+ saturated$ Sample *,00 *cti"ity assay #units$ !000 100 ,00 ,00 ,0 30 -0 50 .0 /0 00 10

.00 ,00

300 ,00

!00 ,00

/5 ,00

50 ,00

-0 !10

!5 !.0

!0 30

2hat is our best option for salting out this protein3 *cti"ity of the protein remains in solution up to .04/0+ ammonium sulfate. So the first step would be to bring the protein solution to /0+ ammonium sulfate to precipitate out the proteins and bring the sample *,00 to -0. 5he protein could then be precipitated out at 10+ ammonium sulfate. 6nce this is done7 the protein would need to be resuspended and the ammonium sulfate should be remo"ed by dialysis. 8ecause we know that the protein has a p% of !07 we know it is a basic protein. %n order to purify this protein7 we should use ion exchange chromatography with a cation exhanger like CM cellulose. Cation e9changers interact with basic proteins #p% !0$. *nion e9changers interact with acidic protein. ,. &escribe the steps to make a monoclonal antibody.

3. * mi9ture of the following amino acids #*la7 Ser7 :he7 ;eu7 *rg7 *sp7 and His$ is sub<ected to paper electrophoresis at pH 3.1. 2hich will go toward the anode3 2hich will go toward the cathode3 *t pH 3.17 what are the charges of the "arious amino acids3 *la =07 Ser =07 :he =07 ;eu =0 but will ha"e a net positi"e charge #some carbo9yl groups will be >C66H but all amino groups will be in ?H3@ state$ thus these will mo"e slightly towards cathode and not separate. His and *rg ha"e p%s closer to /.. and !0.0 and would mo"e towards the cathode7 separating from the other amino acids. *sp with a p% close to 3.0 will mo"e towards the anode. *mino acids with identical charges often separate slightly during paper electrophoresisA for e9ample (ly separates from ;eu. Can you suggest an e9planation3 *lthough identically charged7 a large molecule will mo"e more slowly than a smaller one with the same charge during electrophoresis because the charge4to4mass ratio is smaller and accordingly the force causing migration is smaller per unit mass.

*ssume you ha"e a mi9ture of *la7 Bal7 (lu7 ;ys7 and 5hr at pH ..0. &raw the pattern that will be obtained by ninhydrin staining of the amino acids following paper electrophoresis. %ndicate the anode7 cathode7 origin and any unresol"ed amino acids. * comparison of p%Cs indicates that (lu7 with a net negati"e charge7 will mo"e toward the anodeA ;ys7 with a net positi"e charge7 will mo"e toward the cathode. *t pH =.7 Bal7 *la7 and 5hr are near their isoelectric points. 5hough 5hr might be e9pected to mo"e away from Bal and *la7 it does not do so in practice. #see below$

-. 2hat chromatographic method would be suitable for separating the following pairs of substances3 (a) A-F-K, A-A-K 5hese two differ in the central residue. Since :he is less polar than *la7 paper chromatography can effecti"ely separate these tripeptides. (b) lyso yme, ribonuclease A Since the p%Cs of lysoDyme #p%=!!.0$ and ribonuclease #p% = /.0$ differ so much7 cation e9change chromatography at a pH between these p%Cs would separate these proteins. For e9ample7 CM-cellulose chromatography at p! ".#. (c) hemoglobin, myoglobin Hemoglobe is .-.5 k& and myoglobin is !..1 k& and differ significantly in molecular mass. 5hus gel $iltration chromatography on a gel with a fractionation range will work. Sephade9es (4507 (4!007 (4,007 or 8io4(els :4!07 :4307 :4!007 or Sepharose .b #see 5able .43$ will work. 5. 2hat is the order of elution of the following proteins from a Sephade9 (450 column) catalase7 4chymotrypsin7 concana"alin 87 lipase7 and myoglobin3 5his resin will fractionate based on siDe from ! to 30 k& with the largest eluting first. *ccording to the masses in 5able .457 the order would be) Catalase (%%% k&)'conca(alin ) (*%.+ kd)' -chymotrypsin (%,.- k&)'myoglobin (,-.. k&)'lipase (-./ k&) .. E9plain why the molecular mass of fibrinogen is significantly o"erestimated when

measured using a calibrated gel filtration column #Fig .4!0 in your book$ but can be determined with reasonable accuracy from its electrophoretic mobility on an S&S4polyacrylamide gel #see 5able .45$. 5able .45 indicates that fibrinogen with a fFf0 =,.33.7 is a highly asymmetric molecule. %n its nati"e state it will be more likely to penetrate a gi"en gel pore than a spherical molecule of the same molecular mass. Gnder gel filtration it will migrate more rapidly than this e'ui"alent spherical molecule and hence appear to ha"e a molecular mass lower than it really has. %n contrast7 S&S4:*(E denatures fibrinogen so it will migrate at the same rate as almost any other protein of its molecular mass