bioCHEM48017Practice questions #3; ProtSep.Not to be handed in
(a) If a mixture of the following proteins, collagen (6.6), hemoglobin(7.1), lysozyme(11), albumin(5), and cytochrome c (10.6)were applied to a polystyrene sulfonate ~~~~SO43- exchange chromatography column with a mobile phase of a phosphate buffer @ pH6.0, what would be the order of elution (first on left, last on right)? VVPp99Explain your choice of order……
The stat. phase is –vely charged so most neg proteins will elute first and vice versa.
The higher the pI the more RKH in protein with + ve charges (think histones) so the more overall + , the lower the pI the more DE in protein so overall more –ve.
Albumin / Collagen / Hb / Cyto c / Lysozyme(b) Two of these will probably co-elute. Which and what other technique could be used to possibly separate these two? Explain your choice!Close pIs
Collagen and Hb ORLyso and cyto c)
How experimentally would you be able to tell when the proteins came off the column and the relative amount of each?UV (peak area) or post column derivatization with Comm Blue (VIS) peak areas are proportional to [protein]
2. The mobility of a pure protein, CIA, at pH 8.2 in PAGE is shown below in A. After treatment with SDS and subsequent analysis via SDS/PAGE is shown in B.
When ME was added to the SDS/PAGE, three bands were observed as shown in C.What conclusion can you make about the structure of the pure original protein?
A: PAGE: one sub unit (OR more than one with the same net charge (or same pI see above)
B; SDS/PAGE: two subunits with differing RMM
C: ME/SDS/PAGE: one subunit (larger) has no SS bonds (no change in position in gel) but the other (smaller) has two domains of different RMM linked by SS bonds.Really the two bands from the smaller subunit should be further to the left as they will be smaller than the ‘parent’(as shown above)
Thus CIA is a multi-subunit protein. The two subunits have the same pI but are of different RMM. The smaller subunit has two domains linked by SS bonds.
- You want to separate protein A away from proteins B&C. using only chromatography. Come up with a viable scheme using just 2 columns to accomplish this goal.
Protein / pI / RMM kDa
A / 4.2 / 40
B / 4.4 / 70
C / 8.0 / 44
First IXC or PAGE to separate C from A&B. Collect A&B mixture and use SEC or SDS/Page to separate A&B. (Or the other way round, SEC first then IXC)
- Consider these 5 ‘native’proteins below
(a)Which protein would elute (i) LAST (ii) FIRST from a SEC column? (i) C smallest RMM) (ii) B largest)
(b)Which would migrate the (i) slowest (ii) fastest in SDS-PAGE? (i) ? (i) B largest RMM) (ii) C (smallest)
(c)
(c ) Which protein would elute (i) LAST (ii) FIRST from an anionic exchange (+++ charged) @ pH6.5 column? (i) Last: most neg=lowestpI(i) =D(ii) First: most +ve, highestpI=A
(d)What is the likely quaternary structure for proteins A, B, C, D, & E on the basis of this data?A= tetramer B=Hexamer C=monomer (no 4o) D=trimer E= dimer
(e)What experimental technique could be used to determine the size and thus number of sub units in A, B D, & E?SDS/PAGE and ME/SDS/PAGE
(f)If one used a SEC column with e ‘working’ range of 40-100kDa, how many peaks would you see?First peak B, then second D, third E & fourth A&D together,
Prot / Native Mass (kDa) / Sub Unit Mass (kDa) / pIA / 40 / 10 / 8.1
B / 120 / 15 / 5.5
C / 20 / 20 / 7.2
D / 75 / 25 / 4.5
E / 60 / 30 / 6.2