Question

Pre-Lab 7: Buffers for Cytochrome c purification and redox assays

Describe how to prepare the following solutions in water:

2mL of 45% of ammonium sulfate

50 mL of 20mM Tris pH 8.0 using a 1M Tris pH 8.0 stock solution

50 mL of 1M Glycine pH 10.0

Molecular weight:

Ammonium sulfate = 132.14 g/mol

Tris base = 121.14 g/mol

Glycine = 75.07 g/mol

What is ammonium sulfate precipitation?

What is the isoelectric point (pI) of Cytochrome c?

Answer 1

a) 2mL of 45% ammonium sulphate.

molar mass: 132.14g/mol

45% means 45g in 100 ml of solution. so according to the unitary method, 2mL would need 0.9g of solute. so that means 0.9g of ammonium sulfate is required to prepare its 45% solution.

b)50mL of 20mM Tris from 1M Tris

so according to the principle of conservation of moles, we have S1V1=S2V2

S1= 1M of Tris

V1 is the unknown volume of 1M Tris required to prepare 20mM of Tris.

S2= 20mM of Tris

V2= 50mL

so according to principle, 5020mM= 1M V1

V1= 1mL of 1M Tris

molarity = moles/volume(L)

1M= moles/ 1mL= 1000 moles/1L, so moles = 0.001

mass= moles molar mass =0.001121.14g/mol= 0.12114g is required

c) 50 mL of 1M glycine

molarity =moles/volume(L)

1M= moles/50mL = 1000 moles/50 = 20moles

0.05 moles are required.

mass= moles molar mass = 0.0575.07g/mol = 3.7535 g is required. later the pH is adjusted by adding dilute NaOH in the mixture.

d) The solubility of globular proteins increases upon the addition of ammonium sulfate salt, an effect termed salting-in. At higher salt concentrations, protein solubility usually decreases, leading to precipitation; this effect is termed salting-out. Salts that reduce the solubility of proteins also tend to enhance the stability of the native conformation. In contrast, salting-in ions are usually denaturants. on adding salt in a protein medium, it forms an ionic cage around the protein and thereby stabilizing it by canceling the net charge over protein molecules. thus repulsion between molecules get decreased and they form an aggregate and eventually precipitate down.

e) isoelectric point is a pH value where the whole molecule is overall uncharged/ neutral.

the isoelectric point of cytochrome c is 9.6.