Protein Purification
[1] The Isoelectric Point of Histones
Lehninger chapter 3, page 111, question 13
Show Answer
See answer in Lehninger on page AS–4.
[2] Calculate the Isoelectric Point of a Histone
Calculate the isoelectric point of human histone H1. The
FASTA formatted sequence is available along with the
Protein Composition Tool and
Protein Isoelectric Point Calculator to help with this. Do you results match what is suggested in question 1?
Show Answer
The calculated isoelectric point of human histone H1 is 11.5. Remember that the calculated pI is ±1 pH unit. This is in good agreement with the suggested pI in Leningher. There are three other human histones and a myriad of characterized histones from other organisms; the Lehninger value is a good estimate from all these data.
[3] Purification of an Enzyme
Lehninger chapter 3, page 111, question 15
Show Answer
See answer in Lehninger on page AS–4.
[4] Chromatographic Methods
Lehninger chapter 3, page 112, question 22
Show Answer
See answer in Lehninger on page AS–4.
[5] Purification Tables
Complete the following purification table for the purification of tomato fruit phosphofructokinase (FK) from Martinez–Barajas et al. (1997).
Table 1: Summary of purification of tomato FK expressed in Escherichia coli.
| Fraction |
Total Activity
(μmol*min-1) |
Yield
(%) |
Total Protein
(mg) |
Specific Activity
(μmol*min-1*mg) |
Purification
(fold) |
| Crude |
4.5 |
100 |
31.35 |
|
1 |
| (NH4)2SO4 |
4.0 |
|
8.29 |
|
|
| Mono Q |
3.4 |
|
0.36 |
|
|
| ATP–agarose |
2.5 |
|
0.11 |
|
|
Show Answer
Table 2 below shows the completed purification table, calculated values are in bold. Yield is calculated by dividing the total activity following a purification step by the total activity in the crude homogenate. Specific activity is calculated by dividing the total activity following a step by the total protein following the same step. The fold purification is calculated by dividing the specific activity following a step by the specific activity of the crude homogenate.
Table 2: Summary of purification of tomato FK expressed in Escherichia coli (completed).
| Fraction |
Total Activity
(μmol*min-1) |
Yield
(%) |
Total Protein
(mg) |
Specific Activity
(μmol*min-1*mg) |
Purification
(fold) |
| Crude |
4.5 |
100 |
31.35 |
0.14 |
1 |
| (NH4)2SO4 |
4.0 |
89 |
8.29 |
0.48 |
3.4 |
| Mono Q |
3.4 |
76 |
0.36 |
9.4 |
67 |
| ATP–agarose |
2.5 |
56 |
0.11 |
23 |
160 |
[6] Purification of Human Superoxide Dismutase
Suggest a brief protocol for the purification of human superoxide dismutase by ion exchange chromatography. For your protocol, suggest an ion exchange resin and a pH value at which the enzyme will bind to the resin. The
FASTA formatted sequence of superoxide dimustase is available from NCBI.
Show Answer
The isoelectric point of human superoxide dismutase is 5.7. At pH values below 5.7, the protein has a net positive charge and will bind to a cation exchange resin such as CM cellulose. At pH values above 5.7, the protein has a net negative charge and will bind to an anion exchange resin such as DEAE cellulose.
