To observe the catalytic activity of pepsin (in cleaving peptide bonds in hemoglobin) in the presence and absence of pepstatin. This data will be compared with data we take in a later lab on pepsin-AuNPs.
The experimental details (pepsin cleavage of hemoglobin) are similar to this reference.
We will allow the pepsin cleavage of hemoglobin to proceed for two hours with aliquots taken and analyzed every 1/2 hour. We will also run parallel reactions with different concentrations of pepstatin (pepsin inhibitor) present. We will save our aliquots for analysis tomorrow using SDS-PAGE.
- Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM).
- Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 20 nM, 0.2uM, 2uM, and 20uM)
- Incubate these solutions at 37C (in the incubator shaker)
- After 30 minutes, remove two samples, as instructed below, for analysis today and tomorrow.
- Prepare a sample for SDS-PAGE tomorrow
- Remove 10uL of the reaction sample and dilute to 1mL with the Glycine-HCl buffer
- Take 10uL of this diluted sample and mix with 10uL of the SDS-PAGE running buffer
- Store all of your SDS-PAGE samples in the fridge overnight.
- Prepare a sample for UV-Vis analysis today
- Remove 0.75mL of the reaction sample and add 0.75mL of 1.7M perchloric acid to precipitate the remaining hemoglobin
- After this sample sits for 1 hour, centrifuge for 15 minutes (organize centrifuge time with the other groups) in order to remove solid (uncleaved hemoglobin) from solution
- Measure the absorption spectrum (specifically note 280nm) in order to determine the protein concentration in solution. Use the stock hemoglobin solution as your reference
- Repeat Step 3 every 30 minutes for 2 hours.
Making the solutions
Glycine-HCl Buffer pH 3
- 0.7556g glycine in 200mL water adjust the pH with HCl ---> 50mM Gly-HCl
50 mL of 5 mg/mL Hemoglobin
- 0.2549g hemoglobin in 50mL of Gly-HCl buffer pH 3 ---> 51.0 mg/mL
Pepstatin is not very soluble in water. Solubilize in methanol as per wikipedia.
4.0mg pepstatin in 5mL methanol ---> 1.2mM pepstatin
SDS-PAGE Sample Buffer (No DTT)
- 3.75mL 0.5M Tris pH 6.8 ---> 6.1042 g Tris in 100mL water ---> 0.504M Tris
- 15.0mL of 50% glycerol ---> 7.5mL glycerol + 7.5mL water
- 0.3ml of 1% Bromphenol Blue ---> 0.009g Bromphenol Blue in 1mL water
- 6.0mL 10% Sodium Dodecyl Sulfate (SDS) ---> 1g SDS in 10mL water
- H2O to make 30mL total
4.2mg pepsin in 10mL (MW of pepsin is 34620 according to sigma) ---> 1.2uM
The class ran out of some solutions and Karlena made new:
250.1 mg hemoglobin in 50mL water
Glycine HCl buffer
0.37910g glycine in 100mL water and adjust the pH to 3 ---> 50.5mM Glycine-HCl buffer pH 3
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