Materials:

Equipments:

1. Spectrofluorometer
2. Weighing balance
3. pH meter

Reagents:

1. Urea
2. 3-(N-Morpholino)propanesulfonic acid sodium salt (MOPS sodium salt)
3. 1 M Hydrochloric acid
4. Given protein (RNase T1, commercially available)

Glassware and plasticware:

1. Pipettes
2. Pipette tips
3. 100 ml volumetric flasks
4. 100 ml beaker
5. Test tubes or 15 ml polypropylene tubes
6. Quartz cuvettes

 

Preparation of reagents:

Urea stock solution : Urea stock solution is prepared as follows:

1. Take a 100 ml volumetric flask, place it on the weighing balance, allow the reading to stabilize, and then tare it.
   
   
2. Weigh accurately 60 g of urea and 0.694 g of MOPS sodium salt and add them to a 100 ml beaker. For the sake of doing calculations is step 7, let us assume that the weight of the urea was 59.95 g.
   
   
3. Add 1.8 ml of 1 M HCl and 45 ml of distilled water and allow the urea and MOPS salt to dissolve.
   
   
4. Measure the pH of the solution; if required, adjust the pH using 1 M HCl (note down the added mass).
   
   
5. Transfer the contents of the beaker into the ‘tared' 100 ml volumetric flask.
   
   
6. Add distilled water to make the final volume to 100 ml and weigh the volumetric flask. Let us assume that the weight is 115.07 g.
   
7. Calculate the urea concentration as follows:
1. Calculate the ratio, , let us call this ratio, W .
   Here,
   
   
2. If d is the density of the solution and d₀ is the density of water, then
   
   
   
3. The volume of the solution,
   
   
4. Therefore, the molarity of urea
8. This gives a 9.95 M urea solution in 30 mM MOPS buffer, pH 7.0.

MOPS buffer :

1. Weigh 0.694 g of MOPS sodium salt and transfer to a 100 ml beaker.
2. Add 90 ml of distilled water and allow the salt to dissolve completely.
3. Adjust the pH to 7.0 using 1 N HCl.
4. Transfer the contents to a 100 ml volumetric flask and add distilled water to make the final volume 100 ml .

Protein stock solution :

1. Weigh accurately 100 mg of RNase T1 in a 15 ml polypropylene tube.
2. Add 10 ml of 30 mM MOPS buffer, pH 7.0.
3. This gives a 10 mg/ml solution of RNase T1 in 30 mM MOPS buffer, pH 7.0.
4. Store the protein stock solution at –20 °C.