1. U2P6 and 7 Separation of Macromolecules

2. Your Task Each pair will be given a mixture of three proteins. This mixture will be unique to your group. Your mixture will contain between 4 and 8 mg of any three of the following proteins: Myoglobin, Haemoglobin, Cytochrome c,  -lactalbumin, Ribonuclease, Bovine Serum Albumin, Ferritin and Catalase. It is your task to separate and identify these three proteins.

3. Clues to help you Your mixture of three proteins will either contain 2 heavy and 1 light protein or 1 heavy and 2 light proteins. For the purposes of this experiment, a heavy protein is defined as one with a molecular weight of over 50,000 and a light protein is one with a molecular weight of less than 50,000. The two smaller/heavier proteins in the mixture have pI’s that differ by at least 2 pH units. These can be separated by ion exchange chromatography at pH 7.5.

4. Hint….. Ion Exchange Chromatography involves a charged column, in our case a positively charged matrix, DEAE cellulose. If you can separate 2 proteins by ion exchange chromatography at pH 7.5 then the 2 proteins must have pIs on either side of 7.5 so they are opposite charges at pH 7.5. In this way one protein sticks to the column and the other is eluted.

5. Estimating the charge of a protein [H+] [OH-] [H+] pI ~5 Protein becomes increasingly -ve Protein becomes increasingly +ve [OH-]

6. Estimating the charge of a protein [H+] [OH-] [H+] Protein 1 pI ~5 Protein 1 -ve Protein 1 +ve [OH-] Protein 2 pI ~9 Protein 2 +veProtein 2 -ve

7. Estimating the charge of a protein [H+] [OH-] [H+] Protein 1 pI ~5 Protein 1 -ve Protein 1 +ve [OH-] Protein 2 pI ~9 Protein 2 +veProtein 2 -ve pH of column buffer

8. Before coming to class… Set up a table similar to the one on page 2 of the lab notes. Use the information at the back of these prac notes to fill in the details. For example: Bovine Serum Albumin: This is the most abundant serum protein. It is involved in osmotic regulation and in the transport of metabolites (usually fairly insoluble ones such as fatty acids). It has a molecular weight of 69,000 and an isoelectric point of 4.7. At the pH of blood (7.4) it has a net negative charge of 18. It does not have a visible spectrum and it has an E1% of 6.9 at 280 nm. A410/A280 = 0

9. Before coming to class… Bovine Serum Albumin: This is the most abundant serum protein. It is involved in osmotic regulation and in the transport of metabolites (usually fairly insoluble ones such as fatty acids). It has a molecular weight of 69,000 and an isoelectric point of 4.7. At the pH of blood (7.4) it has a net negative charge of 18. It does not have a visible spectrum and it has an E1% of 6.9 at 280 nm. A410/A280 = 0 ProteinMol. Wt. Native/ subunit pICharge at pH 7.5 Charge at pH 8.8 Absorbance 410:A280 BSA69 000 1 subunit 4.7negative Colourless so no A410

10. Before coming to class… Group the heavy proteins together (those with mol. wts > 50,000) in your table then the light proteins. From this information you should be able to work out whether you have 2 heavy and 1 light protein or 2 light and 1 heavy protein.